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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 methyl
agents 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 not
bromide, also, to a great extent, agricultural. There is                  irreversibly alter neuronal synaptic responses (Zeise
consensus 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 bromide
E-mail address: mzeise@usach.cl (M.L. Zeise).                             does not exert any immediate toxic effect on synaptic

0161-813X/$ – 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) 817–823

transmission in rat hippocampal slices. These and other          constant rate (2.0 ml/min). The composition of the
data, like the prolonged delay between acute exposure            aCSF was (mM): NaCl, 130; KCl 3.5; NaH2PO4
to monohalomethanes and the appearing of clinical                1.25; MgSO4 1.5; CaCl2 2.0; NaHCO3 24; and glucose
signs, 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 Cultures
glutathione by gluthathione S-transferases (GSTs) is
the 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 Stoppini
1983). In mammals, four cytosolic isoforms for this              et al. (1991). Seven days old male Sprague–Dawley
enzyme have been described, called alpha, mu, pi and             rats were anesthetized by hypothermia and rapidly
theta. The theta isoform has been shown to be the one            decapitated. Their brains were quickly removed and
that 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 sterile
tant 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 slices
the hypothesis that SMC could be an essential factor for         (400 mm thick) were obtained using a mechanical tissue
the neurotoxicity of monohalomethanes, either as an              chopper (Stoelting, Kiel, WI). Hipppocampal slices
important 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 left
slices of the rat hippocampus to investigate short-term          on ice for 1–3 h. Then, the slices were placed on
effects, 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 sterile
leave synaptic connections largely intact, unlike cell           collagen solution. The inserts were put into a six-well
cultures, 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 were
the possibility that SMC may act via GABAA receptors             cultured for 3–4 days at 36.5 8C, 100% humidity, in a
by 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 3–4
Freshly 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 Neurons
anesthetized with ethyl ether and decapitated. The
brain 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 maintained
O2) artificial cerebrospinal fluid (aCSF) and used for             in vitro for 12–17 days on 35 mm tissue culture dishes
recording 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 medium
a 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                                         819

SMC Application

   SMC (Sigma, St. Louis, MO) was dissolved in aCSF
and applied by adding it to the perfusion fluid of freshly
prepared slices. After 30 min of exposure, perfusion
was switched back to control solution.
   In organotypic cultures, 2 weeks after dissection,
SMC was added to the culture medium used for regular
replacement. Twenty-four hours later, cultures were
either transferred to the recording chamber perfused
with aCSF devoid of SMC, or they were kept in their
wells and PI was added for the evaluation of cell
membrane integrity (see below).

Electrophysiology

Extracellular Field Recordings
  A bipolar platinum electrode was lowered until it just
touched the slice in the area of the Schaffer collaterals.
Application of rectangular electrical pulses (100 ms,
100–3000 mA) elicited field population spikes that
were recorded from the CA1 pyramidal cell layer using
glass electrodes filled with 2 M NaCl. By the mean of
input/output relationships, stimulus intensities were
determined that yielded the maximal differences
between control and SMC responses (typically just
below the one that elicited maximum response in
control; Fig. 1A). Population spike amplitudes were
measured as the average of 10 repetitive stimulation              Fig. 1. Effect of SMC on population spike amplitudes in freshly
responses. 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 six
more than 10% for three consecutive trials, measure-
                                                                  experiments corresponding to six different slices stemming from at
ments were taken as baseline set to 100% in the                   least three different animals (error bars: S.E.M.). SMC was added
evaluation (see Fig. 1). Slices that displayed visible            to the perfusion fluid (arrow) for 30 min immediately after the third
damage, repetitive discharges in control or population            baseline measurement. (A) Dose–response curves in control and
spike 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. No
tures were performed as described above using a                   significant differences were reached at any stimulus intensity
special recording chamber that allowed the placement              (control: n ¼ 7; SMC: n ¼ 9; error bars: S.E.M.) (B) Application
of 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 holding
glucose. The internal solution contained 120 mM                   potential was À60 mV. Electrodes were pulled from
CsCl, 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 filtered
30 min before starting the recordings. The whole-cell             at 2 kHz and stored for off-line analysis on a PC
recordings were done using an Axopatch-1D amplifier                interfaced with a TL-1 board (Axon Instruments).
820                                       F. Pancetti et al. / NeuroToxicology 25 (2004) 817–823

Propidium 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 averaged
cells only when membranes are severely damaged and                       population spike amplitude Æ S:E:M: between groups
becomes brightly red fluorescent after binding to                         was analyzed using one-way ANOVA. Significance
nucleic acids (Macklis and Madison, 1990). In orga-                      was determined using Tukey’s test with a level of
notypic cultures, after 24 h of exposure to SMC or                       significance set at P < 0:05. For the data obtained
under control conditions, 5 mg/ml PI was added to each                   with 10 Â 10À3 M SMC, recovery data were compared
well together with control culture medium. Three hours                   with the same data set at baseline and application time
later, pictures of the cultures were taken using a                       using Student’s paired t-test. Electrophysiologic data
fluorescence microscope (Olympus BX-50, NIKON,                            obtained from organotypic cultures were evaluated
Melbeach, NY). Determinations were repeated three                        using the one-way ANOVA test.
times and performed in duplicate (using two wells
under the same conditions).                                                                        RESULTS

Statistical 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 both




Fig. 2. Fluorescence caused by PI uptake into hippocampal cells. Organotypic cultures from 1-week-old rat pups, kept in culture for 2 weeks
and 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                                     821

curves did not reach significance at any point, the
maximal difference was consistently detected at stimu-
lus intensities that just induced maximal control
responses. The absolute maximum was not altered by
SMC (Fig. 1A). As shown in Fig. 1B, while SMC at
1 mM caused no detectable change, at higher concen-
trations, it reduced the synaptic population spike in
CA1 pyramidal cells (24:6 Æ 4:3 % at 10À2 M 30 min
after start of application). We observed no sign of a
toxic effect in these experiments, including no repetitive
discharges or changes in the time course of the field
response. There was a highly significant recovery, but it
may not have been complete (the wash out data failed to
be significantly different from the control). There was
                                                                     Fig. 3. Modulation of responses to synaptic stimulation in
no difference in response in control and SMC-treated
                                                                     organotypic cultures (every trace represents the average of three
slices when stimulus repetition frequency was increased              measurements). Left column: responses at the beginning of
from 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, at
5 Â 10À6 and 2 Â 10À5 M, there was no specific signal                 was repeated continuously, using a stimulus repetition
of fluorescence employing PI. However, at 5 Â 10À5 M                  frequency of 0.05 Hz during 10 min, responses of
SMC, we detected damage to the integrity of the cell                 slices that had been exposed to SMC (5 Â 10À6 and
membranes as revealed by the PI uptake. The signal at                2 Â 10À5 M) increased in amplitude while the response
5 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 were
being 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 spike
tions 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 could
synaptic responses were significantly altered. (No                    be detected with repetitive stimulation in control, but at
electrophysiologic 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 to
sumably 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 reduced
spike amplitude was enhanced and repetitive dis-                     GABA-induced currents with an IC50 of almost
charges 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 the
GABA-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 were
without 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, SMC
find serious impairment of electrical properties in               appeared to act as a competitive GABAA receptor
hippocampal neurons exposed to methyl bromide up                 antagonist with an IC50 of 4:4 Â 10À4 M (Fig. 4). This
to millimolar concentrations (Zeise et al., 1999). Thus,         value is very compatible with our fluorescence data
in the present investigation we examined the effects of          (extensive damage at 5 Â 10À4 M), but does not seem
SMC, that is one of the first metabolites appearing in            to explain the electrophysiologic data obtained with
the 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 significant
outside 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 the
or transported into it after having been synthesized in          results of the brain slice study where a reduction of
the 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 of
SMC lowers excitability to synaptic stimulation. This            GABA-induced currents in brain slices, augmenting
effect attains statistical significance only at a concen-         synaptically induced field responses, while, in the
tration of 10À2 M. It is not impossible that this value          whole cell clamp measurements, this effect was quite
may be reached in an accident involving heavy expo-              clear? It may be speculated that, since organotypic
sure and may cause sedation in the person intoxicated.           cultures and dissociated cells were taken from newborn
However, concentrations measured from blood serum                or embryonic animals, while freshly prepared slices
in a serious accident (Garnier et al., 1996) were con-           were from almost adult specimens, these different
siderably 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 region
was 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 as
toxicity. The mechanism of this reduction of field                low as 0.05 Hz mentioned above, may also be caused
response 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 neurons
ciated hippocampal cells SMC acted like a competitive            is subject to frequency dependency causing inhibitory
antagonist (see below).                                          postsynaptic potentials to decrease when stimulus
   A very different picture appeared when we exposed             repetition frequencies exceed 0.1 Hz (Deisz and
organotypic cultures to SMC at medium micromolar                 Prince, 1989). This phenomenon is crucial for the
concentrations for 24 h. This resulted in membrane               induction of LTP (Mott and Lewis, 1991). SMC might
damage as revealed by the intake of PI (5 Â 10À5 M               shift the frequency for the induction of potentiation by
and 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 concentration
amidal 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. Concentrations
rodents this structure matures only in the postnatal             of 5 Â 10À6 to 5 Â 10À5 M, however, that altered
period (Bayer and Altman, 1974), leaving it more                 synaptic responses in organotypic cultures after 24 h
susceptible to the actions of neurotoxic substances.             of exposure are likely to occur in exposed people. In a
More specifically, some GST isoforms are expressed                previous work, we determined bromide level in the
only 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 not
typic cultures at 5 Â 10À6 and 2 Â 10À5 M SMC (an                             ¨
                                                                 exposed (Muller et al., 1999). This corresponds to about
increase 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 medium
run. The effect could be explained by a decrease in              micromolar range and above. Thus, we interpret
GABergic 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                                        823

hippocampal neurons to SMC even at very high con-                     Deisz RA, Prince DA. Frequency-dependent depression of
centrations 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 glutathione
applied for 24 h alter the responses in a way that is                    S-transferases and their significance to toxicology. Toxicol Sci
compatible 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 bromide
substance 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. Macromolecular
necessary for the generation of SMC at least from                        adducts in the use of methyl bromide as fumigant. Toxicol Lett
methyl 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 in
enzymes. 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 Res
brain 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 Appl
clarify 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 propidium
observed 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-term
wagen, 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—a
0075PV 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
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Pancetti et al 2004 methilene

  • 1. 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 methyl agents 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 not bromide, also, to a great extent, agricultural. There is irreversibly alter neuronal synaptic responses (Zeise consensus 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 bromide E-mail address: mzeise@usach.cl (M.L. Zeise). does not exert any immediate toxic effect on synaptic 0161-813X/$ – see front matter # 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2004.01.008
  • 2. 818 F. Pancetti et al. / NeuroToxicology 25 (2004) 817–823 transmission in rat hippocampal slices. These and other constant rate (2.0 ml/min). The composition of the data, like the prolonged delay between acute exposure aCSF was (mM): NaCl, 130; KCl 3.5; NaH2PO4 to monohalomethanes and the appearing of clinical 1.25; MgSO4 1.5; CaCl2 2.0; NaHCO3 24; and glucose signs, 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 Cultures glutathione by gluthathione S-transferases (GSTs) is the 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 Stoppini 1983). In mammals, four cytosolic isoforms for this et al. (1991). Seven days old male Sprague–Dawley enzyme have been described, called alpha, mu, pi and rats were anesthetized by hypothermia and rapidly theta. The theta isoform has been shown to be the one decapitated. Their brains were quickly removed and that 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 sterile tant 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 slices the hypothesis that SMC could be an essential factor for (400 mm thick) were obtained using a mechanical tissue the neurotoxicity of monohalomethanes, either as an chopper (Stoelting, Kiel, WI). Hipppocampal slices important 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 left slices of the rat hippocampus to investigate short-term on ice for 1–3 h. Then, the slices were placed on effects, 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 sterile leave synaptic connections largely intact, unlike cell collagen solution. The inserts were put into a six-well cultures, 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 were the possibility that SMC may act via GABAA receptors cultured for 3–4 days at 36.5 8C, 100% humidity, in a by 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 3–4 Freshly 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 Neurons anesthetized with ethyl ether and decapitated. The brain 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 maintained O2) artificial cerebrospinal fluid (aCSF) and used for in vitro for 12–17 days on 35 mm tissue culture dishes recording 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 medium a 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.
  • 3. F. Pancetti et al. / NeuroToxicology 25 (2004) 817–823 819 SMC Application SMC (Sigma, St. Louis, MO) was dissolved in aCSF and applied by adding it to the perfusion fluid of freshly prepared slices. After 30 min of exposure, perfusion was switched back to control solution. In organotypic cultures, 2 weeks after dissection, SMC was added to the culture medium used for regular replacement. Twenty-four hours later, cultures were either transferred to the recording chamber perfused with aCSF devoid of SMC, or they were kept in their wells and PI was added for the evaluation of cell membrane integrity (see below). Electrophysiology Extracellular Field Recordings A bipolar platinum electrode was lowered until it just touched the slice in the area of the Schaffer collaterals. Application of rectangular electrical pulses (100 ms, 100–3000 mA) elicited field population spikes that were recorded from the CA1 pyramidal cell layer using glass electrodes filled with 2 M NaCl. By the mean of input/output relationships, stimulus intensities were determined that yielded the maximal differences between control and SMC responses (typically just below the one that elicited maximum response in control; Fig. 1A). Population spike amplitudes were measured as the average of 10 repetitive stimulation Fig. 1. Effect of SMC on population spike amplitudes in freshly responses. 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 six more than 10% for three consecutive trials, measure- experiments corresponding to six different slices stemming from at ments were taken as baseline set to 100% in the least three different animals (error bars: S.E.M.). SMC was added evaluation (see Fig. 1). Slices that displayed visible to the perfusion fluid (arrow) for 30 min immediately after the third damage, repetitive discharges in control or population baseline measurement. (A) Dose–response curves in control and spike 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. No tures were performed as described above using a significant differences were reached at any stimulus intensity special recording chamber that allowed the placement (control: n ¼ 7; SMC: n ¼ 9; error bars: S.E.M.) (B) Application of 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 holding glucose. The internal solution contained 120 mM potential was À60 mV. Electrodes were pulled from CsCl, 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 filtered 30 min before starting the recordings. The whole-cell at 2 kHz and stored for off-line analysis on a PC recordings were done using an Axopatch-1D amplifier interfaced with a TL-1 board (Axon Instruments).
  • 4. 820 F. Pancetti et al. / NeuroToxicology 25 (2004) 817–823 Propidium 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 averaged cells only when membranes are severely damaged and population spike amplitude Æ S:E:M: between groups becomes brightly red fluorescent after binding to was analyzed using one-way ANOVA. Significance nucleic acids (Macklis and Madison, 1990). In orga- was determined using Tukey’s test with a level of notypic cultures, after 24 h of exposure to SMC or significance set at P < 0:05. For the data obtained under control conditions, 5 mg/ml PI was added to each with 10 Â 10À3 M SMC, recovery data were compared well together with control culture medium. Three hours with the same data set at baseline and application time later, pictures of the cultures were taken using a using Student’s paired t-test. Electrophysiologic data fluorescence microscope (Olympus BX-50, NIKON, obtained from organotypic cultures were evaluated Melbeach, NY). Determinations were repeated three using the one-way ANOVA test. times and performed in duplicate (using two wells under the same conditions). RESULTS Statistical 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 both Fig. 2. Fluorescence caused by PI uptake into hippocampal cells. Organotypic cultures from 1-week-old rat pups, kept in culture for 2 weeks and exposed to SMC during the last 24 h (DG: dentate gyrus). Two more assays, performed in duplicate, yielded qualitatively similar results.
  • 5. F. Pancetti et al. / NeuroToxicology 25 (2004) 817–823 821 curves did not reach significance at any point, the maximal difference was consistently detected at stimu- lus intensities that just induced maximal control responses. The absolute maximum was not altered by SMC (Fig. 1A). As shown in Fig. 1B, while SMC at 1 mM caused no detectable change, at higher concen- trations, it reduced the synaptic population spike in CA1 pyramidal cells (24:6 Æ 4:3 % at 10À2 M 30 min after start of application). We observed no sign of a toxic effect in these experiments, including no repetitive discharges or changes in the time course of the field response. There was a highly significant recovery, but it may not have been complete (the wash out data failed to be significantly different from the control). There was Fig. 3. Modulation of responses to synaptic stimulation in no difference in response in control and SMC-treated organotypic cultures (every trace represents the average of three slices when stimulus repetition frequency was increased measurements). Left column: responses at the beginning of from 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, at 5 Â 10À6 and 2 Â 10À5 M, there was no specific signal was repeated continuously, using a stimulus repetition of fluorescence employing PI. However, at 5 Â 10À5 M frequency of 0.05 Hz during 10 min, responses of SMC, we detected damage to the integrity of the cell slices that had been exposed to SMC (5 Â 10À6 and membranes as revealed by the PI uptake. The signal at 2 Â 10À5 M) increased in amplitude while the response 5 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 were being 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 spike tions 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 could synaptic responses were significantly altered. (No be detected with repetitive stimulation in control, but at electrophysiologic 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 to sumably 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 reduced spike amplitude was enhanced and repetitive dis- GABA-induced currents with an IC50 of almost charges 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 the GABA-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 were without effect (not shown).
  • 6. 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, SMC find serious impairment of electrical properties in appeared to act as a competitive GABAA receptor hippocampal neurons exposed to methyl bromide up antagonist with an IC50 of 4:4 Â 10À4 M (Fig. 4). This to millimolar concentrations (Zeise et al., 1999). Thus, value is very compatible with our fluorescence data in the present investigation we examined the effects of (extensive damage at 5 Â 10À4 M), but does not seem SMC, that is one of the first metabolites appearing in to explain the electrophysiologic data obtained with the 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 significant outside 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 the or transported into it after having been synthesized in results of the brain slice study where a reduction of the 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 of SMC lowers excitability to synaptic stimulation. This GABA-induced currents in brain slices, augmenting effect attains statistical significance only at a concen- synaptically induced field responses, while, in the tration of 10À2 M. It is not impossible that this value whole cell clamp measurements, this effect was quite may be reached in an accident involving heavy expo- clear? It may be speculated that, since organotypic sure and may cause sedation in the person intoxicated. cultures and dissociated cells were taken from newborn However, concentrations measured from blood serum or embryonic animals, while freshly prepared slices in a serious accident (Garnier et al., 1996) were con- were from almost adult specimens, these different siderably 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 region was 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 as toxicity. The mechanism of this reduction of field low as 0.05 Hz mentioned above, may also be caused response 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 neurons ciated hippocampal cells SMC acted like a competitive is subject to frequency dependency causing inhibitory antagonist (see below). postsynaptic potentials to decrease when stimulus A very different picture appeared when we exposed repetition frequencies exceed 0.1 Hz (Deisz and organotypic cultures to SMC at medium micromolar Prince, 1989). This phenomenon is crucial for the concentrations for 24 h. This resulted in membrane induction of LTP (Mott and Lewis, 1991). SMC might damage as revealed by the intake of PI (5 Â 10À5 M shift the frequency for the induction of potentiation by and 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 concentration amidal 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. Concentrations rodents this structure matures only in the postnatal of 5 Â 10À6 to 5 Â 10À5 M, however, that altered period (Bayer and Altman, 1974), leaving it more synaptic responses in organotypic cultures after 24 h susceptible to the actions of neurotoxic substances. of exposure are likely to occur in exposed people. In a More specifically, some GST isoforms are expressed previous work, we determined bromide level in the only 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 not typic cultures at 5 Â 10À6 and 2 Â 10À5 M SMC (an ¨ exposed (Muller et al., 1999). This corresponds to about increase 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 medium run. The effect could be explained by a decrease in micromolar range and above. Thus, we interpret GABergic inhibition, since in dissociated hippocampal our results in such a way that short-term exposure of
  • 7. F. Pancetti et al. / NeuroToxicology 25 (2004) 817–823 823 hippocampal neurons to SMC even at very high con- Deisz RA, Prince DA. Frequency-dependent depression of centrations 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 glutathione applied for 24 h alter the responses in a way that is S-transferases and their significance to toxicology. Toxicol Sci compatible 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 bromide substance 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. Macromolecular necessary for the generation of SMC at least from adducts in the use of methyl bromide as fumigant. Toxicol Lett methyl 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 in enzymes. 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 Res brain 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 Appl clarify 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 propidium observed 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-term wagen, 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—a 0075PV 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 in Bayer 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.