Toxicology in Vitro xxx (2009) xxx–xxx

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Toxicology in Vitro

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In vitro assessment of paraoxon effects on GABA uptakein rat hippocampal synaptosomes

Fereshteh Pourabdolhossein a, Asghar Ghasemi b,*, Ameneh Shahroukhi c, Mohammad Amin Sherafat a,Ali Khoshbaten d, Alireza Asgari d

a Department of Physiology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iranb Endocrine Physiology Laboratory, Endocrine Research Center, Research Institute for Endocrine Sciences, Shaheed Beheshti University (M.C.), P.O. Box 19395-4763, Tehran, Iranc District Health Center of Shahre Ray, Tehran University of Medical Sciences, Tehran, Irand Department of Physiology and Biophysics and Research Center for Chemical Injuries, Baqiyatallah University of Medical Sciences, Tehran, Iran

a r t i c l e i n f o

Article history:Received 27 January 2009Accepted 10 May 2009Available online xxxx

Keywords:GABA uptakeHippocampusOrganophosphateParaoxonSynaptosome

0887-2333/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.tiv.2009.05.003

* Corresponding author. Tel.: +98 21 22432500; faxE-mail address: Ghasemi@endocrine.ac.ir (A. Ghas

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a b s t r a c t

Treating organophosphate poisoning is achieved mainly using compounds with anticholinergic charac-teristics. Nevertheless currently the focus of attention is aimed at examining their interference with otherneurotransmitter systems. The present investigation studied the potential interactions between parao-xon and GABA uptake in hippocampal synaptosomes. Wistar rats weighing 200–250 g were used. Hippo-campal synaptosomes were prepared and incubated with [3H] GABA in the presence of different doses ofparaoxon for 10 min at 37 �C; and were then layered in chambers of a superfusion system and the [3H]GABA uptake was measured. Our finding revealed that mean GABA uptake decreased by 21%, 42%, 37%,20%, and 8% of the corresponding control values in the presence of paraoxon concentrations of 0.01,0.1, 1, 10, and 100 lM, respectively which was significant at 0.1 and 1 lM of paraoxon (P < 0.05). In con-clusion, micromolar concentrations of paraoxon were shown to interfere with GABA uptake in hippocam-pal synaptosomes, which indicates the GABA transporters may play a role in organophosphate-inducedconvulsions.

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1. Introduction

As agricultural and house insecticides, organophosphates (OPs)are in wide use around the world, and a significant number of poi-sonings and deaths are reported each year as a result of exposureto them (London et al., 2005). Being one of the most toxic OP pes-ticides, parathion, whose active metabolite is paraoxon (O,O-diethyl p-nitrophenyl phosphate), has a major contribution tooccupational and accidental intoxication, consequences of its com-mon use in agriculture (Vatanparast et al., 2006).

A great deal of evidence demonstrates that OPs have directinteractions with other molecular targets or cellular functions inthe CNS, such as membrane channels (Rocha et al., 1996), pumps(Dierkes-Tizek et al., 1984), receptors (McDonough and Shih,1997), and even neurotransmitters (McDonough and Shih, 1997).In addition, it has been confirmed that paraoxon causes morpho-logical changes in neurons (Yousefpour et al., 2006). As a resultof the mentioned mechanisms, OPs induce seizure in hippocampusand cortex of brain (Mohammadi et al., 2008).

Irreversible inhibition of acetylcholinesterase (AChE) is re-garded as the main mechanism of OPs, resulting in accumulation

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: +98 21 22416264.emi).

sein, F., et al. In vitro assessme5.003

of acetylcholine (ACh) in cholinergic synapses and consequentoverstimulation of cholinergic system (Moser, 1995; Rocha et al.,1996). The excess ACh stimulates seizure activity (convulsions)which if produced in susceptible areas and, if uncontrolled, pro-gresses rapidly to status epilepticus, causing profound neural dam-age (Shih et al., 1999). Despite the above-mentioned mechanismcontributing largely to seizure activity, not all signs of OP poison-ing can be attributed to it. Therapeutic drugs used to treat OP-in-duced convulsion mainly antagonize the cholinergic effects ofOPs and do not eliminate all symptoms, implying that accompa-nied by cholinergic system, other neurotransmission systemsmay be involved (Capacio and Shih, 1991). Given that seizure activ-ity of OP poisoning is mostly attributed to the GABAergic system, itseems to be the principal candidate to be investigated. A change inthe levels of GABA during nerve agent seizures has been reported(Kar and Matin, 1972; McDonough and Shih, 1997). Furthermore,an increase in inhibitory activity of the GABAergic system has beenreported for almost all drugs that have beneficial effects againstOP-induced convulsions (Shih et al., 1999). Enhancement of the re-lease of GABA, reduction in its uptake (by different known trans-porters) or both mechanisms are the existing evidence for theinvolvement of GABA system in OP poisoning. It has been demon-strated that the number of GABA transporters decreases in epilep-tic tissues (Bradford, 1995). Being the major determinant of the

nt of paraoxon effects on GABA uptake in rat hippocampal synaptosomes.

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level of tonic inhibition and a significant source of GABA releaseduring seizures, GABA transporter plays a much more dynamic rolein the control of brain excitability than documented previously(Richerson and Wu, 2004). Currently there are limited reports spe-cifically on the effect of OPs on GABA uptake; two recent in vitroinvestigations carried out in our laboratory indicated that parao-xon is capable of inhibiting tritiated GABA uptake by cerebellar(Shahroukhi et al., 2007) and cortical (Ghasemi et al., 2007) synap-tosomes in rats. Similar results have been obtained in an in vivostudy in which synaptosomes were prepared from paraoxon-trea-ted rats (Mohammadi et al., 2008). Synaptosomes are very usefulto study different synaptic events including uptake (Garcia-Sanzet al., 2001).

Due to its central role in the initiation and maintenance of epi-leptic seizures, the hippocampus is an area of particular interest.Moreover, this structure has a vast cholinergic innervation (Lewiset al., 1967) and high number of GABA transporters, GAT1 (Cecchi-ni et al., 2004). Owing to its ability to exhibit epileptiform activity,together with variety of manipulations that can induce this state,in vitro study of the hippocampus is widely used to investigate sei-zure activity and evaluate antiepileptic/anticonvulsant drugs (Oli-ver et al., 1977).

The present in vitro study was performed with the aim of deter-mining the effect of paraoxon on GABA uptake and acetylcholines-terase activity in synaptosomes prepared from the untreated-rathippocampus, for its potential involvement in seizure attacks andto facilitate novel strategies for the treatment of OP poisoning.

2. Experimental procedures

2.1. Chemicals

[3H] GABA (86 Ci/mmol) was purchased from Amersham Biosci-ence UK. Diethyl paraoxon, nipecotic acid, aminooxyacetic acid,gamma-aminobutyric acid (GABA), and acetylthiocholine (ATC)were obtained from the Sigma Chemical Co., Germany. 5,50-dithi-obis 2-nitrobenzoic acid (DTNB) and bovine serum albumin (BSA)from Fluka (Swiss); other materials were provided by the MerckCompany, Germany.

2.2. Animals

Male Wistar rats (200–250 g) were kept in 22 ± 2 �C and 12 h/12 h light-dark cycles having access to food and water ad libitium,during the course of study. All animal experiments were conductedaccording to the established protocols of the Ethical Committee ofthe Baqiyatallah Medical Sciences University.

2.3. Synaptosome preparation

Synaptosomes were prepared as previously described by Raiteriet al. (2003). In brief, hippocampi (0.6 ± 0.05 g) were dissected andhomogenized in 0.32 M sucrose buffered with 100 mM phosphate,pH 7.4. In order to remove cell debris, the homogenate was centri-fuged 5 min at 1000g. The supernatant was then centrifuged at12000g for 20 min. The pellets, containing synaptosomes, werethen resuspended in buffer solution containing (in mM): NaCl125, KCl 3, MgSO4 1.2, CaCl2 1.2, NaHCO3 22, NaH2PO4 1, pH 7.4,saturated with 95% O2 and 5% CO2. The protein concentrationwas adjusted to 1 mg/ml.

2.4. Biochemical assays

Protein concentration was determined by Bradford’s method(Bradford, 1976), BSA being used as the standard. Lactate dehydro-

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genase (LDH) activity, as biochemical proof for synaptosomalmembrane integrity, was assessed with reduction of pyruvate tolactate (Moss and Henderson, 1994). Synaptosomal LDH activitywas measured in the presence (total activity) and absence (freeactivity) of 1% Triton X-100 and the occluded activity was ex-pressed as percent of total. Acetylcholinesterase activity was deter-mined by the Ellman method as previously described by Dietz et al.(1973). In brief, synaptosomes were solublized in Triton X-100. Theincubation mixture with a total volume of 2.55 ml, consisted of:2 ml DTNB (0.37 mM) in 0.1 M phosphate buffer (pH 7.6), 0.45 mlsynaptosome homogenate, and 0.1 ml of ATC (13.5 mM). Absor-bance at 410 nm was read every 30 second for 5 min and cholines-terase activity calculated as nanomoles substrate hydrolyzed permin per mg protein.

2.5. [3H] GABA uptake assay

Uptake assays were performed according to the Mantz andcoworkers method (Mantz et al., 1995). The synaptosomal pelletswere resuspended to a final protein concentration of 1 mg/ml inbuffer solution. Aliquots of synaptosomes (0.5 ml, containing0.5 mg protein) were added to tubes, preincubated for 20 min at37 �C in either the absence (control) or presence of various concen-trations of paraoxon (0.01, 0.1, 1, 10, and 100 lM) and then incu-bated with 400 nM GABA (1.5% of which was tritiated) at 37 �Cfor 10 min (10 min, because this time was in the linear portion oftime-dependent [3H] GABA uptake). Each concentration was as-sayed in triplicate in four independent experiments. In addition,0.5 ml of each synaptosomal preparation was added to the tubecontaining the GABA uptake inhibitor nipecotic acid (final concen-tration, 50 mM). Aminooxyacetic acid (10 lM) was used in allexperiments to prevent GABA catabolism.

After incubation (10 min), the reaction was stopped by additionof 2 ml of ice-cold buffer solution and synaptosomes were washedthree times with ice-cold buffer solution after transfer to superfu-sion chambers. The synaptosomes were filtered onto 0.65 lm poreMillipore filters, placed at the bottom of a set of parallel superfu-sion chambers (Raiteri and Raiteri, 2000). A peristaltic pump wasconnected to the bottom of the superfusion chambers and its flowadjusted to 0.5 ml/min. The filters, containing the remnant of syn-aptosomes, were completely covered by scintillated liquid andtheir radioactivities were counted with a liquid scintillation coun-ter (Betamatic, Kontron, France). Specific GABA uptake was calcu-lated as total uptake minus uptake in presence of 50 mMnipecotic acid (an inhibitor of GABA uptake). A few experimentswere done in which sodium chloride was substituted by lithiumchloride (equimolar) to clarify transporter-dependency of uptake.

2.6. Statistical analysis

The results were presented as mean ±S.E.M. Comparison ofGABA uptake and AChE activity was done by one-way analysis ofvariance (ANOVA), and if ANOVA results were significant, multiplecomparisons followed by post hoc Tukey test. Two-sided P-valuesless than 0.05 were considered significant. Data analyses weredone by the SPSS program (version 9).

3. Results

3.1. Biochemical analyses

We used changes in occluded LDH activity as a marker for theintegrity of rat hippocampal synaptosomes (Fig. 1). Disruption ofparticles with Triton X-100 increased enzyme activity significantly.When expressed as a percentage of the total, occluded and free

nt of paraoxon effects on GABA uptake in rat hippocampal synaptosomes.

*

0

10

20

30

40

50

60

70

80

Occluded Free

LDH

act

ivity

(% o

f con

trol)

Fig. 1. Occluded and free LDH activity (percent of total), of rat hippocampalsynaptosomes. Results of mean ± SE for 18 experiments (*P < 0.05).

-4 -3 -2 -1 0 1 2

0

20

40

60

80

100

120

Log [paraoxon] µM

Cho

lines

tera

se a

ctiv

ity(%

of

cont

rol)

Fig. 2. Synaptosomal cholinesterase inhibition by paraoxon. 50% inhibitory con-centration of paraoxon (IC50) is 24.8 nM.

*

0

20

40

60

80

100

NaCl LiCl

[3 H]G

ABA

upta

ke (%

)

Fig. 3. Effect of substituting lithium chloride for sodium chloride on [3H] GABAuptake by rat brain synaptosome. Data demonstrated that GABA uptake wassodium dependent (*P < 0.001).

Fig. 4. Time-dependency of GABA uptake. It reaches a maximum at 20 min afterbeginning of incubation.

**

0

20

40

60

80

100

0 0.01 0.1 1 10 100[Paraoxon] µM

[3H

]GA

BA

upt

ake

(% o

f con

trol)

Fig. 5. Effect of paraoxon on [3H] GABA uptake in rat hippocampal synaptosomes.Values are expressed as the percentage of the control uptake (*P < 0.05 compared tothe control group).

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LDH activities were 78 ± 1% and 21 ± 1%, respectively (P < 0.05,n = 18). Paraoxon inhibited cholinesterase activity of synapto-somes, in a concentration-dependent manner. Fifty percent inhib-itory concentration of Paraoxon (IC50) for cholinesterase inhibitionwas 24.8 nM (n = 4 in triplicate) (Fig. 2).

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3.2. Sodium dependency of uptake

When lithium chloride was substituted for sodium chloride,GABA uptake decreased significantly to 21% of the initial value(P < 0.001, n = 8) (Fig. 3).

3.3. Time-dependency of [3H] GABA uptake

Synaptosomal [3H] GABA uptake was time-dependent andpeaked at 20 min after incubation. [3H] GABA uptake were 30,63, 85, 100 and 100 pmol/mg protein at 5, 10, 15, 20, and 30 minafter beginning of incubation. We selected 10 min as an appropri-ate incubation time in order to indicate more precisely the GABAuptake changes (Fig. 4).

3.4. Effect of paraoxon on [3H] GABA uptake

Paraoxon decreased the [3H] GABA uptake, which was signifi-cant at 0.1 and 1 lM (P < 0.05, n = 8). No significant differencewas observed between other paraoxon concentrations (Fig. 5).The inhibitory effect was attenuated at 10 lM and at higher dosesof paraoxon. Nipecotic acid (50 mM) caused 95% inhibition inGABA uptake, which indicates that most of the GABA uptake occursvia transporters.

nt of paraoxon effects on GABA uptake in rat hippocampal synaptosomes.

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3.5. Cholinergic-independency of paroxon on [3H] GABA uptake

We studied GABA uptake at 1 and 3 lM concentrations of ace-tylcholine (doses which were higher than the concentrationneeded to inhibit acetylcholinesterase activity) in order to investi-gate the cholinergic-independency of this uptake in the presence ofparaoxon. The results indicated that acetylcholine has no effect onGABA uptake, verification of GABA uptake being cholinergic-inde-pendent in the presence of paraoxon (n = 6, Fig. 6).

3.6. Nipecotic acid

With the aim of studying GABA uptake in the presence of parao-xon, we made use of nipecotic acid as an inhibitor of GABA uptake;[3H] GABA uptake in the presence of 5, 10, and 50 mM nipecoticacid concentrations was 16%, 12%, and 3% of control group, respec-tively (P < 0.001, n = 8) (Fig. 7).

0

20

40

60

80

100

0 1 3[Acetylcholine] µM

GAB

A up

take

(% o

f con

trol)

Fig. 6. Cholinergic-independency of paroxon on GABA uptake. No effect wasobserved at acetylcholine concentrations of 1 and 3 lM on GABA uptake in rathippocampal synaptosomes.

***0

20

40

60

80

100

Control 5 10 50[Nipecotic acid] mM

% [3 H

]GAB

A up

take

Fig. 7. Inhibition of [3H] GABA uptake in rat hippocampal synaptosomes bynipecotic acid. *P < 0.001.

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4. Discussion

In our study, cholinesterase was inhibited by paraoxon in a con-centration-dependent manner. In addition, we showed that micro-molar concentrations of paraoxon interfere with GABAtransmission and reduce its uptake in rat hippocampal synapto-somes. This is a concentration that seems to be sufficient to induceseizure (Rocha et al., 1996). Hippocampus was selected for its keyrole in the initiation and maintenance of epileptic seizures(Mohammadi et al., 2008) and high number of GAT1 transporters(Cecchini et al., 2004).

Decreases in GABA uptake observed in this study are consistentwith our previous studies on cerebral and hippocampal synapto-somes prepared from paraoxon-treated rats (Mohammadi et al.,2008) and on the effect of millimolar concentration of paraoxonon GABA uptake in rat cerebral cortex synaptosomes (Ghasemiet al., 2007). This effect may be related to the decrease in maxi-mum velocity of uptake by GABA transporters (Bahena-Trujilloand Arias-Montano, 1999). In a previous study on the cerebral cor-tex synaptosomes we found that micromolar concentrations ofparaoxon increase GABA uptake (Ghasemi et al., 2007), a findingcontradictory to the results of the present study. An explanationfor this discrepancy may be related to different IC50 of paraoxonfor inhibiting cholinesterase activity, which was approximatelythree times greater in hippocampal synaptosomes compared tocerebral cortex ones. Although high levels of GAT-1 mRNA havebeen found in cortex and hippocampus (Dalby, 2003; Cecchiniet al., 2004) difference in the number of transporters may be theanother reason for this inconsistency.

GABA has a significant degree of extracellular freedom follow-ing release. Removing GABA from the extra-synaptic space duringboth low- and high-frequency firing is carried out through GABAtransport (Kinney and Spain, 2002); thus these transporters regu-late synaptic and extra-synaptic concentrations of GABA and, inthis capacity, are partly responsible for the regulation of inhibitoryneurotransmission in the nervous system (Whitlow et al., 2003).GABA transporters may have other important functions as well,such as supplying a source of extracellular GABA via reversal ofthe GABA transporter and regulating paracrine GABA (Wu et al.,2001; Kinney and Spain, 2002). Under physiological conditions, asmall increase in cellular GABA (5–10%) may be sufficient to stim-ulate GABA transporter reversal activity (Wu et al., 2001). Inhibi-tion of GABA uptake by paraoxon decreases the synaptic contentof GABA, favoring reduction in GABA release. In other words, it ispossible that inhibition of the GABA transporter causes the de-crease in GABA release mediated by transporters, contributing tothe seizure activity by paraoxon. This would only occur if existingGABA diffuses out of its microenvironment, a phenomenon re-ported previously (Kinney and Spain, 2002).

The stoichiometry of GABA transporters has been reported to be1GABA:2Na+:1Cl� (Lu and Hilgemann, 1999). Considering GABAtransport is Na+-dependent, our experiments showed that equimo-lar substitution of Na+ with Li+ significantly attenuated uptake of[3H] GABA which indicates Na+-dependency of uptake (Ghasemiet al., 2007) as reported by others (Lu and Hilgemann, 1999). Forthe mechanism of the inhibitory effect of paraoxon on GABA up-take, it is interesting to consider that GABA transporter has Na+�

dependency. So the direct toxic inhibitory effect of OPs on so-dium-potassium ATPase (Dierkes-Tizek et al., 1984; Blasiak 1995;Karalliedde 1999) leads to disruption of the concentrations of Na+

and may interfere with GABA transport. Through this mechanism,the effects of OPs on GABA uptake may presumably be justified.

To provide evidence for the accuracy of our results, we exam-ined our synaptosomes functionally. The membrane integrity ofsynaptosomes was therefore confirmed by both LDH and nipecoticacid experiments. Previously we showed that similar values of LDH

nt of paraoxon effects on GABA uptake in rat hippocampal synaptosomes.

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activity before and after paraoxon exposure indicate that paraoxondid not disrupt membrane integrity (Ghasemi et al., 2007), as re-ported by Cecchini et al. (Cecchini et al., 2004). It was observed thatthe optimum time for synaptosomal [3H] GABA uptake was about10 min, reaching a maximum at 20 min, which is consistent withthat reported by Sutch and coworkers, who claimed [3H] GABA up-take was maximum at 20 min (Sutch et al., 1999). This incubationtime does not disrupt synaptosomes as determined by measuringthe LDH activity before and after incubation.

Paraoxon leads to seizure by influencing the mechanisms con-cerning acetylcholine and GABA. In our study, acetylcholine couldnot alter the effect of paraoxon on GABA uptake, suggesting thatthis effect is cholinergic-independent, although the same dose ofparaoxon completely inhibited cholinesterase (IC50 = 24.8 nM).

Redistribution of GABA transporters between cell surface andcytoplasm is considered a regulatory mechanism for transportersfunction (Szilagyi et al., 1993). Hence, it may be suggested thatdecreasing GABA uptake is performed by paraoxon via transporterinternalization.

During intoxication, the concentration of paraoxon in the bloodcan temporarily reach the micromolar concentration range (DeNeef et al., 1983); yet it is quite possible that cell function in thetarget tissues may be altered by exposure to much lower concen-trations of OPs. Although not measured in our in vitro study, parao-xon concentration in the brain may reach levels of 10 lM duringclinical intoxication (Rocha et al., 1996).

In the present study, paraxon inhibited GABA uptake with max-imum inhibition observed at 0.1 lM. Based on statistical analyses,significant differences were observed only in 0.1 lM and 1 lMconcentrations. This may be justified by the speculation that athigher concentrations, paraoxon exerts its effect through alterna-tive processes, and paraoxon concentration-dependency of GABAtransporters is most probably the potential cause of this phenom-enon. Nevertheless, the exact reason why high concentrations ofparaoxon are less effective on GABA uptake remains to be eluci-dated. In a quite different design of experiment, Rocha and col-leagues (Rocha et al., 1996) reported that submicromolarconcentrations of paraoxon increased the frequency of miniaturepostsynaptic currents, whilst the reverse occurred at millimolarconcentrations. In addition, working on the effect of cannabinoidson GABA uptake by rat brain slices, Vendora and coworkers dem-onstrated a bell-shaped relationship (Venderova et al., 2005). Allthe findings mentioned can be put into a category that supportsthe notion of concentration-dependent behavior of GABA uptakein the presence of paraoxon. It is probable that this concentra-tion-dependent effect may be the underlying reason for the exist-ing controversial reports on GABA levels following OP intoxication(Kar and Matin, 1972; Coudray-Lucas et al., 1984; Fosbraey et al.,1990). From another perspective, the contradictions in the litera-ture may be attributed to the fact that, neurotransmitter releasein synaptic terminals is a complex phenomenon and is modulatedat several putative sites, including the transporter as well as the re-lease process itself (Larsson et al., 1986). To resolve current con-tradictions, we recommend a more in-depth investigation of theintercellular networks composed of the processes contributing tothe toxic effect of OPs. To summarize, the present results suggestthat micromolar concentrations of paraoxon decrease [3H] GABAuptake in synaptosomal preparation. As a result, transporter func-tion is positively correlated with neurotransmitter release andGABA transporters may play a role in OP-induced convulsion.

Acknowledgement

This work was supported by the Research Center for ChemicalInjuries of the Baqiyatallah University of Medical Sciences andTarbiat Modares University, to which we forward our deep appre-

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ciations. We thank Mrs. B. Soleymani for technical assistance andMrs. N. Shiva for linguistic help.

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nt of paraoxon effects on GABA uptake in rat hippocampal synaptosomes.