NeuroToxicology 25 (2004) 817–823 S-Methylcysteine may be a Causal Factor in Monohalomethane Neurotoxicity Floria Pancetti1,2, Marcelo Oyarce1, Marcela Aranda2, Jorge Parodi3, Luis G. Aguayo3, Bernardo Morales2, Gotz Westphal4, Michael Muller4, Ernst Hallier4, Marc L. Zeise1,* ¨ ¨ 1 Department of Management in Agriculture, Technological Faculty, University of Santiago de Chile, Santiago, Chile 2 Department of Biological Sciences, Faculty of Chemistry and Biology, University of Santiago de Chile, Santiago, Chile 3 ´ ´ Laboratory of Neurophysiology, Department of Physiology, University of Concepcion, Concepcion, Chile 4 ¨ Department of Occupational and Social Medicine, Georg-August-University, Gottingen, Germany Received 3 July 2003; accepted 10 January 2004 Available online 12 March 2004 Abstract S-Methylcysteine (SMC) is formed after exposure to monohalomethanes in rodents as well as in humans. The present study was performed to study whether SMC, directly or indirectly, contributes to the well-known neurotoxicity of monohalomethanes. We have investigated the effects of acute exposure to SMC by means of electrophysiolocal measurements in freshly prepared hippocampal slices and dissociated hippocampal neurons in culture. For longer- term exposures (24 h) we have used organotypic cultures (2 weeks in culture), taking electrophysiologic recordings and assessing membrane integrity with propidium iodide (PI) ﬂuorescence. We found that only high concentrations of SMC (10À2 M; exposure time 30 min) in freshly isolated slices of adult rats reduce synaptically evoked population spikes in the CA1 region. This effect was at least partially reversible. In organotypic cultures, at 5 Â 10À5 M after 24 h of exposure, SMC compromises membrane integrity as revealed by PI ﬂuorescence, only in the dentate gyrus, spreading to pyramidal cell layers at 5 Â 10À4 M. At 5 Â 10À6 and 2 Â 10À5 M, under the same experimental conditions, no changes were seen with the PI method, but we recorded increased population spike amplitudes, repetitive discharges and frequency potentiation (at a stimulus repetition rate of 0.05 Hz). Using whole-cell patch clamp in hippocampal dissociated neurons we have found that SMC (applied for approximately 1 s) reduces GABA-induced currents (IC50 ¼ 4:4 Â 10À4 M) without having an effect of its own, acting like a competitive antagonist at GABAA receptors. Our ﬁndings are in line with the view that the ability of monohalomethanes to induce the formation of SMC is an important factor for their neurotoxicity, provided that SMC is allowed to act at least for several hours. The effects exerted by SMC seem to be due, at least in part, to its interaction with GABA receptors. # 2004 Elsevier Inc. All rights reserved. Keywords: S-Methylcysteine; Monohalomethanes; GABA; Hippocampus; Organotypic cultures INTRODUCTION major concern (Alexeef and Kilgore, 1983; Bonnefoi et al., 1991). However, the mechanisms underlying Monohalomethanes are important methylating neurotoxic action of methyl bromide as well as methylagents that represent considerable hazards to human iodide (Chamberlain et al., 1999) are still unclear.health and the earth’s ozone layer (McCauley et al., In a previous work, we have shown that concentra-1999). Their use is industrial and, in the case of methyl tions of up to 5 Â 10À2 M of the bromide ion do notbromide, also, to a great extent, agricultural. There is irreversibly alter neuronal synaptic responses (Zeiseconsensus that neurotoxicity of these compounds is a et al., 1999). Thus, at least in the case of methyl bromide, it is most likely that methylation rather than * Corresponding author. Tel.: þ56-2-682-2520; the halogen anion is the primary cause for its toxicity.fax: þ56-2-682-2521. The same study demonstrated that methyl bromideE-mail address: firstname.lastname@example.org (M.L. Zeise). does not exert any immediate toxic effect on synaptic0161-813X/$ – see front matter # 2004 Elsevier Inc. All rights reserved.doi:10.1016/j.neuro.2004.01.008
818 F. Pancetti et al. / NeuroToxicology 25 (2004) 817–823transmission in rat hippocampal slices. These and other constant rate (2.0 ml/min). The composition of thedata, like the prolonged delay between acute exposure aCSF was (mM): NaCl, 130; KCl 3.5; NaH2PO4to monohalomethanes and the appearing of clinical 1.25; MgSO4 1.5; CaCl2 2.0; NaHCO3 24; and glucosesigns, have led to the hypothesis that toxicity is caused 10; pH 7.4.by metabolites rather than by direct action. Conjuga-tion of the methyl group of monohalomethanes to Organotypic Culturesglutathione by gluthathione S-transferases (GSTs) isthe major pathway for metabolization of this class of Hippocampal slice cultures were prepared by the ¨compounds (Jager et al., 1988; Kornbrust and Bus, interface culture method, as described by Stoppini1983). In mammals, four cytosolic isoforms for this et al. (1991). Seven days old male Sprague–Dawleyenzyme have been described, called alpha, mu, pi and rats were anesthetized by hypothermia and rapidlytheta. The theta isoform has been shown to be the one decapitated. Their brains were quickly removed andthat metabolizes monohalomethanes in human blood placed in ice cold Hank’s Balanced Salt Solution(Guengerich et al., 1995; Sheehan et al., 2001). (HBSS; GIBCO, Langley, OK) supplemented with S-Methylcysteine (SMC) is a quantitatively impor- 28 mM glucose in a 60-mm culture dish under steriletant immediate metabolite resulting from GST activity conditions. Hippocampi were dissected out under a(Goergens et al., 1994). In the present work, we tested stereomicroscope. Transversal hippocampal slicesthe hypothesis that SMC could be an essential factor for (400 mm thick) were obtained using a mechanical tissuethe neurotoxicity of monohalomethanes, either as an chopper (Stoelting, Kiel, WI). Hipppocampal slicesimportant mediator or by being neurotoxic of its own. were carefully transferred to another sterile 60 mm dish We have applied SMC to freshly isolated in vitro containing HBSS supplemented with glucose and leftslices of the rat hippocampus to investigate short-term on ice for 1–3 h. Then, the slices were placed oneffects, and to organotypic cultures for 24 h in order to Millicell-CM sterile tissue culture plate inserts (Milli-study longer-lasting exposure. These preparations pore, Bedford, MA) previously covered with sterileleave synaptic connections largely intact, unlike cell collagen solution. The inserts were put into a six-wellcultures, while allowing for control of concentrations culture tray, each well containing 1 ml of ‘‘optimem’’and other experimental parameters that are not so well culture medium (GIBCO) supplemented with 25%deﬁned in in vivo models. It also permits the detection heat-inactivated horse serum, 25% HBSS, penicillin,of plastic changes in synaptic transmission that may be G/streptomycin sulfate (5000 units/ml and 5 mg/ml,relevant in neurological changes. We further addressed respectively) and 28 mM glucose; pH 7.3. Slices werethe possibility that SMC may act via GABAA receptors cultured for 3–4 days at 36.5 8C, 100% humidity, in aby measuring the inﬂuence of SMC on GABA-induced 95% air–5% CO2 atmosphere. After 3–4 days of incu-currents in dissociated hippocampal cells. bation the culture medium was replaced with 1 ml of chemically deﬁned serum-free neurobasal medium (GIBCO) containing 1 mM L-glutamine, 2% B27 sup- MATERIALS AND METHODS plement (GIBCO) and 28 mM glucose. Thereafter, the medium was changed twice a week during the next 3–4Freshly Isolated Slices weeks. Cultures were regularly checked microscopi- cally. SMC was added with the last culture medium Slices were prepared and maintained as reported exchange and left for 24 h.earlier (Zeise et al., 1992). Brieﬂy, rats (Sprague–Dawley; weighing 80–100 g, about 4 weeks old) were Cultured Hippocampal Neuronsanesthetized with ethyl ether and decapitated. Thebrain was dissected out and transverse hippocampal Hippocampal neurons were dissociated and main-slices (350–400 mm thick) were cut using a Campden tained as described before (Aguayo and Pancetti,vibratome (Campden Instruments, UK). Slices were 1994). Brieﬂy, cells were taken from 18- to 19-day-stored at room temperature in gassed (95% CO2, 5% timed pregnant Sprague–Dawley rats and maintainedO2) artiﬁcial cerebrospinal ﬂuid (aCSF) and used for in vitro for 12–17 days on 35 mm tissue culture dishesrecording no earlier than half an hour and no later than coated with poly(L-lysine). The neuronal feeding med-12 h after preparation. They were singly transferred to ium consisted of 90% minimal essential mediuma recording chamber, completely submerged in and (GIBCO), 10% heat-inactivated horse serum (GIBCO)continuously superfused by the same gassed aCSF at a and a mixture of nutrient supplements.
F. Pancetti et al. / NeuroToxicology 25 (2004) 817–823 819SMC Application SMC (Sigma, St. Louis, MO) was dissolved in aCSFand applied by adding it to the perfusion ﬂuid of freshlyprepared slices. After 30 min of exposure, perfusionwas switched back to control solution. In organotypic cultures, 2 weeks after dissection,SMC was added to the culture medium used for regularreplacement. Twenty-four hours later, cultures wereeither transferred to the recording chamber perfusedwith aCSF devoid of SMC, or they were kept in theirwells and PI was added for the evaluation of cellmembrane integrity (see below).ElectrophysiologyExtracellular Field Recordings A bipolar platinum electrode was lowered until it justtouched the slice in the area of the Schaffer collaterals.Application of rectangular electrical pulses (100 ms,100–3000 mA) elicited ﬁeld population spikes thatwere recorded from the CA1 pyramidal cell layer usingglass electrodes ﬁlled with 2 M NaCl. By the mean ofinput/output relationships, stimulus intensities weredetermined that yielded the maximal differencesbetween control and SMC responses (typically justbelow the one that elicited maximum response incontrol; Fig. 1A). Population spike amplitudes weremeasured as the average of 10 repetitive stimulation Fig. 1. Effect of SMC on population spike amplitudes in freshlyresponses. Only when these values did not deviate isolated brain slices recording from the CA1 soma region. For each concentration of SMC and the control, data were collected from sixmore than 10% for three consecutive trials, measure- experiments corresponding to six different slices stemming from atments were taken as baseline set to 100% in the least three different animals (error bars: S.E.M.). SMC was addedevaluation (see Fig. 1). Slices that displayed visible to the perfusion fluid (arrow) for 30 min immediately after the thirddamage, repetitive discharges in control or population baseline measurement. (A) Dose–response curves in control andspike amplitudes of less than 1 mV were discarded. 10À3 M SMC; measured 30 min after wash in of SMC, 40 min after Electrophysiologic recordings from organotypic cul- the beginning of recording; control: I/O data were measured correspondingly 40 min after the beginning of recording. Notures were performed as described above using a significant differences were reached at any stimulus intensityspecial recording chamber that allowed the placement (control: n ¼ 7; SMC: n ¼ 9; error bars: S.E.M.) (B) Applicationof the plate inserts. of SMC at various concentrations for 30 min: Only the reduction of responses at 10À2 M, measured at the end of the application period,Whole Cell Patch Clamp Recordings is significantly different from control (ANOVA, Tukey’s test; P < 0:05). At this concentration, recovery values just failed to be For whole cell patch clamp recordings, the culture significantly different from the baseline (Student’s paired t-test;dish containing dissociated hippocampal neurons was P ¼ 0:056).continuously perfused with an external solution con-taining 150 mM NaCl, 5.4 mM KCl, 2.0 mM CaCl2,1.0 mM MgCl2, 10 mM HEPES, pH 7.4, and 10 mM (Axon Instruments, Union City, CA). The holdingglucose. The internal solution contained 120 mM potential was À60 mV. Electrodes were pulled fromCsCl, 10 mM BAPTA, 10 mM HEPES, 4 mM MgCl2 borosilicate capillary glass (World Precision Instru-and 2 mM ATP-disodium, pH 7.35. The cells were ments, Sarasota, FL) on a vertical puller (Sutter Instru-stabilized at room temperature (20–22 8C) for at least ments, Novato, CA). The current signals were ﬁltered30 min before starting the recordings. The whole-cell at 2 kHz and stored for off-line analysis on a PCrecordings were done using an Axopatch-1D ampliﬁer interfaced with a TL-1 board (Axon Instruments).
820 F. Pancetti et al. / NeuroToxicology 25 (2004) 817–823Propidium Iodide (PI) Uptake using slices from at least three different animals. The data were analyzed using the program Instat (Graphpad Propidium iodide is a polar compound that enters Software Inc.). The percentage change in averagedcells only when membranes are severely damaged and population spike amplitude Æ S:E:M: between groupsbecomes brightly red ﬂuorescent after binding to was analyzed using one-way ANOVA. Signiﬁcancenucleic acids (Macklis and Madison, 1990). In orga- was determined using Tukey’s test with a level ofnotypic cultures, after 24 h of exposure to SMC or signiﬁcance set at P < 0:05. For the data obtainedunder control conditions, 5 mg/ml PI was added to each with 10 Â 10À3 M SMC, recovery data were comparedwell together with control culture medium. Three hours with the same data set at baseline and application timelater, pictures of the cultures were taken using a using Student’s paired t-test. Electrophysiologic dataﬂuorescence microscope (Olympus BX-50, NIKON, obtained from organotypic cultures were evaluatedMelbeach, NY). Determinations were repeated three using the one-way ANOVA test.times and performed in duplicate (using two wellsunder the same conditions). RESULTSStatistical Analysis Acute effects of SMC were examined by exposing freshly prepared hippocampal slices for half an hour to For the experiments with freshly prepared hippo- low millimolar concentrations. In Fig. 1A, an input/campal slices, the recordings obtained for each group output relationship for control and 10À3 M SMC is(control, 1, 5 and 10 Â 10À3 M SMC) were collected displayed. Even though the difference between bothFig. 2. Fluorescence caused by PI uptake into hippocampal cells. Organotypic cultures from 1-week-old rat pups, kept in culture for 2 weeksand exposed to SMC during the last 24 h (DG: dentate gyrus). Two more assays, performed in duplicate, yielded qualitatively similar results.
F. Pancetti et al. / NeuroToxicology 25 (2004) 817–823 821curves did not reach signiﬁcance at any point, themaximal difference was consistently detected at stimu-lus intensities that just induced maximal controlresponses. The absolute maximum was not altered bySMC (Fig. 1A). As shown in Fig. 1B, while SMC at1 mM caused no detectable change, at higher concen-trations, it reduced the synaptic population spike inCA1 pyramidal cells (24:6 Æ 4:3 % at 10À2 M 30 minafter start of application). We observed no sign of atoxic effect in these experiments, including no repetitivedischarges or changes in the time course of the ﬁeldresponse. There was a highly signiﬁcant recovery, but itmay not have been complete (the wash out data failed tobe signiﬁcantly different from the control). There was Fig. 3. Modulation of responses to synaptic stimulation inno difference in response in control and SMC-treated organotypic cultures (every trace represents the average of threeslices when stimulus repetition frequency was increased measurements). Left column: responses at the beginning offrom 0.2 to 0.5 Hz (not shown). stimulation; right column: responses after 10 min of repetitive In organotypic cultures, that had been maintained in stimulation at 0.05 Hz.culture for 2 weeks and exposed to SMC during 24 h, at5 Â 10À6 and 2 Â 10À5 M, there was no speciﬁc signal was repeated continuously, using a stimulus repetitionof ﬂuorescence employing PI. However, at 5 Â 10À5 M frequency of 0.05 Hz during 10 min, responses ofSMC, we detected damage to the integrity of the cell slices that had been exposed to SMC (5 Â 10À6 andmembranes as revealed by the PI uptake. The signal at 2 Â 10À5 M) increased in amplitude while the response5 Â 10À5 M was conﬁned to the dentate gyrus (identi- amplitude in control cultures was not changed signiﬁ-ﬁed by comparison with Nissl stainings; not shown), cantly (Fig. 3). Qualitatively identical results werebeing more intense and widespread, involving also the obtained in two more controls and two more applica-CA1 region at 5 Â 10À4 M (Fig. 2; n ¼ 3, determina- tions of SMC at either concentration (population spiketions taken in duplicate). amplitudes were always larger with SMC than in As shown in Fig. 3, at 5 Â 10À6 and 2 Â 10À5 M control and no statistically signiﬁcant change couldsynaptic responses were signiﬁcantly altered. (No be detected with repetitive stimulation in control, but atelectrophysiologic recording could be performed in least 50% enhancement with SMC).cultures exposed to SMC 5 Â 10À5 M or more, pre- In dissociated hippocampal cells, SMC, up tosumably due to the damage exerted by SMC as indi- 5 Â 10À3 M, applied alone did not evoke any current.cated by the PI uptake). Firstly, the ﬁeld population However, when co-applied with GABA, SMC reducedspike amplitude was enhanced and repetitive dis- GABA-induced currents with an IC50 of almostcharges were observed. Moreover, when stimulation 5 Â 10À4 M (Fig. 4).Fig. 4. Effect of SMC on GABA-induced currents in dissociated hippocampal cells. SMC, when applied together with GABA, reduces theGABA-induced current in a concentration-dependent manner (IC50 ¼ 4:41 Â 10À4 M þ 70 S.E.M; n ¼ 7). Applications of SMC alone werewithout effect (not shown).
822 F. Pancetti et al. / NeuroToxicology 25 (2004) 817–823 DISCUSSION cells from rat embryos, SMC reduced GABA-induced currents in a concentration-dependent manner being In a previous study (see Introduction), we could not without effect when applied alone. Thus, SMCﬁnd serious impairment of electrical properties in appeared to act as a competitive GABAA receptorhippocampal neurons exposed to methyl bromide up antagonist with an IC50 of 4:4 Â 10À4 M (Fig. 4). Thisto millimolar concentrations (Zeise et al., 1999). Thus, value is very compatible with our ﬂuorescence datain the present investigation we examined the effects of (extensive damage at 5 Â 10À4 M), but does not seemSMC, that is one of the ﬁrst metabolites appearing in to explain the electrophysiologic data obtained withthe brain after monohalomethane exposure. SMC is 5 Â 10À6 and 2 Â 10À5 M, although even minor reduc-generated mainly by mediation of GSTs inside or tion of GABAergic inhibition may cause signiﬁcantoutside of the brain. At the moment, data are lacking modiﬁcations in the behavior of the neuronal circuitry.as to whether SMC present in the brain is formed there There is, however, an apparent contradiction with theor transported into it after having been synthesized in results of the brain slice study where a reduction ofthe blood. ﬁeld responses was observed at high concentrations of Our results show that, at an exposure time of 30 min, SMC (10À2 M). Why did SMC not act as an inhibitor ofSMC lowers excitability to synaptic stimulation. This GABA-induced currents in brain slices, augmentingeffect attains statistical signiﬁcance only at a concen- synaptically induced ﬁeld responses, while, in thetration of 10À2 M. It is not impossible that this value whole cell clamp measurements, this effect was quitemay be reached in an accident involving heavy expo- clear? It may be speculated that, since organotypicsure and may cause sedation in the person intoxicated. cultures and dissociated cells were taken from newbornHowever, concentrations measured from blood serum or embryonic animals, while freshly prepared slicesin a serious accident (Garnier et al., 1996) were con- were from almost adult specimens, these differentsiderably lower. Thus, we may assume that the ‘‘acute results are due to differences in maturation of post-effect’’ of SMC, lowering neuronal excitability, that synaptic responses as described for the CA1 regionwas reversible, at least partially, cannot be considered (Wang et al., 2002).as being of much practical importance related to The frequency potentiation, at a repetition rate astoxicity. The mechanism of this reduction of ﬁeld low as 0.05 Hz mentioned above, may also be causedresponse amplitude is unclear but probably not due by mechanisms involving presynaptic GABAB recep-to increased GABAergic inhibition because in disso- tors since GABAergic inhibition of pyramidal neuronsciated hippocampal cells SMC acted like a competitive is subject to frequency dependency causing inhibitoryantagonist (see below). postsynaptic potentials to decrease when stimulus A very different picture appeared when we exposed repetition frequencies exceed 0.1 Hz (Deisz andorganotypic cultures to SMC at medium micromolar Prince, 1989). This phenomenon is crucial for theconcentrations for 24 h. This resulted in membrane induction of LTP (Mott and Lewis, 1991). SMC mightdamage as revealed by the intake of PI (5 Â 10À5 M shift the frequency for the induction of potentiation byand above; Fig. 2). Interestingly, at 5 Â 10À5 M the PI an interaction with these receptors.signal was strictly limited to the dentate gyrus. Pyr- As mentioned above, we believe that a concentrationamidal cells were affected only at 5 Â 10À4 M. The of 10À2 M SMC in brain tissue will not occur fre-higher vulnerability of the dentate gyrus may have to quently, if ever, rendering low its presumable impor-do with neuronal maturation. It has been shown that in tance in monohalomethane poisoning. Concentrationsrodents this structure matures only in the postnatal of 5 Â 10À6 to 5 Â 10À5 M, however, that alteredperiod (Bayer and Altman, 1974), leaving it more synaptic responses in organotypic cultures after 24 hsusceptible to the actions of neurotoxic substances. of exposure are likely to occur in exposed people. In aMore speciﬁcally, some GST isoforms are expressed previous work, we determined bromide level in theonly in mature tissues (Eaton and Bammler, 1999). blood of workers exposed to methyl bromide to be on The electrophysiologic changes recorded in organo- average about 4 mg/l greater than that of workers nottypic cultures at 5 Â 10À6 and 2 Â 10À5 M SMC (an ¨ exposed (Muller et al., 1999). This corresponds to aboutincrease in population spike amplitude and higher 5 Â 10À5 M of bromide. In individual cases and acci-susceptibility to frequency potentiation) may have dental poisonings, concentrations may be much higher.excitotoxic consequences, particularly in the long This may lead to concentrations of SMC in the mediumrun. The effect could be explained by a decrease in micromolar range and above. Thus, we interpretGABergic inhibition, since in dissociated hippocampal our results in such a way that short-term exposure of
F. Pancetti et al. / NeuroToxicology 25 (2004) 817–823 823hippocampal neurons to SMC even at very high con- Deisz RA, Prince DA. Frequency-dependent depression ofcentrations does not induce toxicity immediately, inhibition in guinea-pig neocortex in vitro by GABAB receptor feed-back on GABA release. J Physiol Lond 1989;412:513–41.whereas medium micromolar concentrations of SMC Eaton DL, Bammler TK. Concise review of the glutathioneapplied for 24 h alter the responses in a way that is S-transferases and their significance to toxicology. Toxicol Scicompatible with toxic effects. 1999;49:156–64. Taken together, our results are compatible with a role ¨ Garnier R, Rambourg-Schepens MO, Muller A, Hallier E.of SMC as an intermediate and/or directly acting Glutathione transferase activity and formation of macromolecular adducts in two cases of acute methyl bromidesubstance that underlies neurotoxicity of monohalo- poisoning. Occup Environ Med 1996;53:211–5.methanes. This also implies a role for GSTs, that are ¨ Goergens HW, Hallier E, Muller A, Bolt HM. Macromolecularnecessary for the generation of SMC at least from adducts in the use of methyl bromide as fumigant. Toxicol Lettmethyl bromide and methyl chloride, whose methylat- 1994;72:199–202.ing power is not strong enough to produce important Guengerich FP, Thier R, Persmark M, Taylor JB, Pemble SE,amounts of SMC without the interference of these Ketterer B. Conjugation of carcinogens by Q class glutathiones S-transferases: mechanisms and relevance to variations inenzymes. However, even in methyl iodide metabolism, human risk. Pharmacogenetics 1995;5:S103–7.GSTs seem to play a major role (Bonnefoi et al., 1991). ¨ Jager R, Peter H, Sterzel W, Bolt HM. Biochemical effects of The role of GSTs in the formation of SMC in the methyl chloride in relation to its tumorigenicity. J Cancer Resbrain should be further investigated as well as the Clin Oncol 1988;114:64–70.interaction of SMC with GABA receptors in order to Kornbrust DJ, Bus JS. The role of glutathione and cytochrome P- 450 in the metabolism of methyl chloride. Toxicol Applclarify whether an excitotoxic action caused by Pharmacol 1983;67:246–56.reduced GABAergic inhibition is essential for the Macklis JD, Madison RD. Progressive incorporation of propidiumobserved neurotoxicity of SMC. iodide in cultured mouse neurons correlates with declining electrophysiological status: a fluorescence scale of membrane integrity. J Neurosci Methods 1990;31:43–6. ACKNOWLEDGEMENTS McCauley SE, Goldstein AH, DePaolo DJ. An isotopic approach for understanding the CH(3)Br budget of the atmosphere. Proc Natl Acad Sci USA 1999;96:10006–9. This study was supported by the Foundation Volks- Mott DD, Lewis DV. Facilitation of the induction of long-termwagen, project no. I/73 050 to MZ; FONDECYT, potentiation by GABAB receptors. Science 1991;252:1718–20.project no. 1030220 to BM and FP; and the University ¨ ¨ Muller M, Reinhold P, Lange M, Zeise M, Jurgens U, Hallier E.of Santiago de Chile through DICYT, project no. Photometric determination of human serum bromide levels—a0075PV to FP and project no. 029971Z to MZ.. convenient biomonitoring parameter for methyl bromide exposure. Toxicol Lett 1999;107:155–9. Sheehan D, Meade G, Foley VM, Dowd CA. Structure, function and evolution of glutathione transferases: implications for REFERENCES classification of non-mammalian members of an ancient enzyme superfamily. Biochem J 2001;360:1–16.Aguayo LG, Pancetti FC. Ethanol modulation of the gamma- ¨ Stoppini L, Buchs P-A, Muller D. A simple method for aminobutyric acid A- and glycine-activated ClÀ current in organotypic cultures of nervous tissue. J Neurosci Methods cultured mouse neurons. J Pharmacol Exp Ther 1994;270:61–9. 1991;37:173–82.Alexeef BV, Kilgore WW. Methylbromide. Residue Rev 1983;88: Wang DS, Inokuchi H, Tanaka E, Isagai T, Li JS, Higashi H. 101–53. Postnatal changes in the overall postsynaptic currents evoked inBayer SA, Altman J. Hippocampal development in the rat: CA1 pyramidal neurons of the rat hippocampus. Life Sci cytogenesis and morphogenesis examined with autoradiography 2002;72:341–53. and low-level X-irradiation. J Comp Neurol 1974;158:55–79. ¨ Zeise ML, Teschemacher A, Arriagada J, Zieglgansberger W.Bonnefoi MS, Davenport CJ, Morgan KT. Metabolism and Corticosterone reduces synaptic inhibition of rat hippocampal and toxicity of methyl iodide in primary dissociated neural cell neocortical neurons in vitro. J Neuroendocrinol 1992;4:107–12. cultures. Neurotoxicology 1991;12:33–46. ´ Zeise ML, Jofre D, Morales P, Espinoza J, Nalli A, Aranda M.Chamberlain MP, Sturgess NC, Lock EA, Reed CJ. Methyl iodide Methyl bromide decreases excitability without having toxicity in rat cerebellar granule cells in vitro: the role of immediate toxic effects in rat hippocampal CA1 neurons in glutathione. Toxicology 1999;139:27–37. vitro. Neurotoxicology 1999;20:827–32.