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Effects of dimethylarsinic and dimethylarsinous acid on evoked synaptic potentials in hippocampal slices of young and adult rats
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Toxicology and Applied Pharmacology 225 (2007) 40 – 46 www.elsevier.com/locate/ytaap
Effects of dimethylarsinic and dimethylarsinous acid on evoked synaptic potentials in hippocampal slices of young and adult rats Katharina Krüger a,⁎,1 , Hendrik Repges a,1 , Jörg Hippler c , Louise M. Hartmann c , Alfred V. Hirner c , Heidrun Straub a , Norbert Binding b , Ulrich Mußhoff a a b
Institut für Physiologie I, Universitätsklinikum Münster, Robert-Koch-Straße 27a, D-48149 Münster, Germany Institut für Arbeitsmedizin, Universitätsklinikum Münster, Robert-Koch-Straße 51, D-48149 Münster, Germany c Institut für Umweltanalytik, Universität Duisburg-Essen, Universitätsstraße 3-5, D-45141 Essen, Germany Received 21 March 2007; revised 6 July 2007; accepted 18 July 2007 Available online 25 July 2007
Abstract In this study, the effects of pentavalent dimethylarsinic acid ((CH3)2AsO(OH); DMAV) and trivalent dimethylarsinous acid ((CH3)2As(OH); DMAIII) on synaptic transmission generated by the excitatory Schaffer collateral-CA1 synapse were tested in hippocampal slices of young (14–21 day-old) and adult (2–4 month-old) rats. Both compounds were applied in concentrations of 1 to 100 μmol/l. DMAV had no effect on the amplitudes of evoked fEPSPs or the induction of LTP recorded from the CA1 dendritic region either in adult or in young rats. However, application of DMAIII significantly reduced the amplitudes of evoked fEPSPs in a concentration-dependent manner with a total depression following application of 100 μmol/l DMAIII in adult and 10 μmol/l DMAIII in young rats. Moreover, DMAIII significantly affected the LTP-induction. Application of 10 μmol/l DMAIII resulted in a complete failure of the postsynaptic potentiation of the fEPSP amplitudes in slices taken both from adult and young rats. The depressant effect was not reversible after a 30-min washout of the DMAIII. In slices of young rats, the depressant effects of DMAIII were more pronounced than in those taken from adult ones. Compared to the (absent) effect of DMAV on synaptic transmission, the trivalent compound possesses a considerably higher neurotoxic potential. © 2007 Elsevier Inc. All rights reserved. Keywords: Hippocampus; Slice; Synaptic transmission; Organoarsenicals; DMA; Long-term potentiation
Introduction Chronic environmental and occupational exposure to arsenic compounds has been associated with numerous adverse health effects, among them lung, skin and other cancers as well as abnormal skin pigmentation, keratosis, hypertension, respiratory disease, gastrointestinal disturbances, anemia and splenomegaly (see, e.g., review, Hall, 2002). Neurological effects of arsenic after occupational or environmental exposure or accidental intoxication, such as subclinical nerve injuries (Lagerkvist and Zetterlund, 1994), delirium and encephalopathy (Morton and Caron, 1989), peripheral neuropathies (Gerr et al., 2000; Mazumder et al., 1992), ⁎ Corresponding author. Institute of Physiology I Robert-Koch-Str. 27a, D48149 Münster, Germany. Fax: +49 251 8355551. E-mail address: [email protected] (K. Krüger). 1 These authors contributed equally to this work. 0041-008X/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2007.07.007
and symptoms including loss of hearing and taste, blurred vision, tingling and numbness of the limbs, and decrease in muscle strength (Liu et al., 2002; Luong and Nguyen, 1999), have been reported. Furthermore, several investigations revealed that arsenicals have an influence on learning and memory. In children, chronic exposure to inorganic arsenic via drinking water resulted in a dose-dependent reduction in intellectual functions (Wasserman et al., 2004; Wright et al., 2006). Alterations in memory and attention have been observed in adolescents after chronic exposure to high levels of arsenic (Tsai et al., 2003). In experimental studies, behavioral alterations have been observed in rats following sodium arsenite treatment (Rodriguez et al., 2001). Effects of arsenite on brain monoamine concentrations have been found in experimental animals (Delgado et al., 2000). While the clinical outcomes of arsenic poisoning are well documented, the toxicological mechanisms involved are still not completely understood. One reason is that inorganic arsenic
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compounds (iA III and iAV ) are rapidly metabolized to methylated pentavalent and trivalent species (monomethylarsonic acid (MMAV ), monomethylarsonous acid (MMA III ), dimethylarsinic acid (DMAV ), and dimethylarsinous acid (DMAIII); Aposhian et al., 2000; Thomas et al., 2004). In most cases, it is not known whether the inorganic compounds are the ultimate toxins or their organic metabolites. Biomethylation of inorganic arsenic has been considered a major detoxification pathway for a long time. This is now questionable, since pentavalent and especially trivalent methylated arsenicals exhibit cytotoxicity (Petrick et al., 2000; Styblo et al., 2000; Cohen et al., 2002) and influence cell proliferation and cytokine secretion in human epidermal keratinocytes (Vega et al., 2001). Viability testing with these keratinocytes showed increasing toxicities of arsenicals in the following order: iAV b MMAV b DMAV b DMAIII b MMAIII b iAIII. From these experimental studies, it is concluded that methylated arsenicals, especially the trivalent species, may significantly contribute to the adverse effects of inorganic arsenite (Styblo et al., 2000; Vahter, 2002). The aim of this study was to investigate a possible contribution of the methylated arsenic metabolites DMAV and DMAIII to the known neurotoxic effects induced by exposure to inorganic arsenite. Hippocampal slices from rat brain were chosen to analyze the effects of pentavalent and trivalent DMA on synaptic transmission at identified synaptic sites. Materials and methods Substances. Dimethylarsinic acid (DMAV) was purchased from Strem (Kehl, Germany). Dimethyliodoarsine (Me2AsI) was kindly donated by Prof. W. Cullen (University of British Columbia, Vancouver, Canada). Preparation of dilute solutions of this iodide precursor results in the formation of the corresponding acid, dimethylarsinous acid (DMAIII) (Gong et al., 2001; Millar et al., 1960). Since only a small amount of DMAIII was available, the measurements with DMAIII were reduced in number. All chemicals were of analytical grade or the highest quality available. In a series of experiments on Xenopus oocytes (Krüger et al., 2006a), the stability of both arsenicals was tested by HPLC-hydride generation atomic fluorescence spectroscopy (HPLC-HG-AFS) using the method of Le et al. (2000). In these experiments, DMAIII and DMAV were added to a modified Ringer's solution which served as diluting agent and was composed of (in mmol/l): NaCl 115, KCl 2, CaCl2 1.8, HEPES 10; pH 7.2. DMAV was found to be stable, i.e., there was no detectable loss in concentration or species transformation over a minimum 2-month period. DMAIII was both volatile and highly susceptible to oxidation; within 7 days less than 20% of the original DMAIII concentration was present and by 30 days no DMAIII could be detected. Around half of the DMAIII losses were accounted for by oxidation of the DMAIII to DMAV, the remainder was presumably volatilized. Storage temperature did not significantly affect the stability of DMAIII. Stock solutions of 1 mmol/L DMAV and DMAIII were prepared in artificial cerebrospinal fluid (ACSF) and stored at 4 °C in glass bottles. The ACSF contained (in mmol/l) NaCl 124, KCl 4, CaCl2 2, NaH2PO4 1.24, NaHCO3 26, MgCl2 1.3 and glucose 10. Due to its high volatility and sensitivity to oxidation, solutions of DMAIII were always prepared immediately before each experiment and discarded if not used within 2 days. Preparation of hippocampal slices. The technique for preparation of hippocampal tissue slices has been described in detail elsewhere (Musshoff et al., 2002). Briefly, 32 adult rats (Wistar, 220–360 g, mean 290 g, 2–4 months old, male) and 25 young rats (Wistar, 25–60 g, mean 39 g, 14–21 days old, mean 17 days old) were decapitated under ether anesthesia. All procedures utilizing animals were performed in accordance with regulations through the German law and were approved by the Bezirksregierung Münster. Both hippocampi were excised from the brain and submerged in chilled (2–4 °C) ACSF for about
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1 min. Slices of 500 μm thickness were cut from the dorsal hippocampus and pre-incubated in ACSF in a submersion chamber for at least 60 min at 28 °C. The ACSF was constantly bubbled with 95% O2 and 5% CO2. For electrophysiological investigations, slices were maintained in a submergedtype recording chamber with the temperature (32 °C), pH (7.4) and flow rate (4 ml/min, bath volume 1 ml) of the ACSF being continuously monitored. Stock solutions of DMAV or DMAIII were diluted immediately prior to experiments and added to the bath solution in concentrations of 1, 10, 25 or 100 μmol/l. Induction of excitatory post-synaptic potentials. fEPSPs were elicited by Schaffer collateral stimulation through a bipolar stimulation electrode and recorded as extracellular field potentials with ACSF-filled glass recording electrodes (0.5–1.5 MΩ) placed in the stratum radiatum of the CA1 region. The synaptic response to a standard test stimulus (0.033 Hz) was monitored until a stable recording was obtained, and the input–output relationship was then determined. The stimulus strength (0.2–2.5 mA) producing a response of approximately 50% of the maximal response amplitude was determined and used for all subsequent experiments. Only synaptic potentials with more than 0.2 mV and without superimposed population spikes were used for the experiments. After stable baseline recording of the responses for 20 min, DMAV or DMAIII was bath-applied to the hippocampal slices for 60 min and then washed out for a further 30 min. The evoked synaptic responses were recorded every 30 s during bathapplication of DMAV or DMAIII and for a further 30 min after washout. Induction of long-term potentiation (LTP). The baseline presynaptic stimulation was delivered at 0.033 Hz for 20 min using a stimulation intensity evoking approximately 50% of the maximal post-synaptic responses. Following the application of a high-frequency stimulus (HFS: 100 Hz, 1 s duration), stimulation was again delivered at 0.033 Hz but now resulted in a potentiated synaptic response which was recorded for a further 90 min. DMAV or DMAIII was bath-applied to the hippocampal slices 15 min before LTP induction and washed out 15 min after setting the high-frequency stimulus. Analyses. The evoked synaptic responses were recorded and analyzed with a personal computer using custom-developed software (LTP program, version 2.30D; Anderson and Collingridge, 2001) and a digital oscilloscope. The fEPSPs were quantified by measurements of the amplitude of the synaptic responses. Each of the amplitudes of the fEPSPs obtained during DMA application was normalized to the average amplitude of the 10 min baseline recordings of the fEPSPs acquired before DMAV or DMAIII application. For LTP experiments, the amplitudes of the post-tetanic responses were normalized to the average of 10-min baseline responses obtained before setting the LTP stimulus. The significance of the differences between the means was calculated for different points in time (t = 15, 30, 60, 90 min), using a t-test or Mann–Whitney rank sum test. Values were considered significantly different if p ≤ 0.05. In the text, values are shown as mean ± S.E.M.
Results fEPSP control measurements To test whether the organoarsenic compounds affect excitatory transmission at the Schaffer collateral-CA1 synapse, evoked fEPSPs were measured under control conditions (ACSF) and after administration of the organoarsenic compounds in ACSF. Under control conditions, the amplitudes of the fEPSPs remained stable compared to the baseline reference over the whole range of 90 min. The mean fEPSP amplitudes and SEM are listed in Table 1 for different points in time and, 15 min after the HFS, were 100% in adult rats (Figs. 1b, 2b, n=6) and 103% in young rats (Figs. 1d, 2d, n=7). Effects of DMAV on fEPSP Original recordings with and without bath application of 100 μmol/l DMAV are shown in Fig. 1a for adult and Fig. 1c for young rats. Bath application of 100 μmol/l DMAV had no
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Table 1 Mean fEPSP amplitudes ± SEM compared to the baseline reference fEPSP
Adult rats
Point in time CTRL 100 μmol/l DMAV 1 μmol/l DMAIII 10 μmol/l DMAIII 100 μmol/l DMAIII
t = 15 100 ±3% 96 ±3% 98 ±2% 90 ±1% 45 ±2%
Young rats t = 30 100 ±3% 96 ±3% 91 ±4% 72 ±3% 9 ±1%
t = 60 106 ±3% 101 ±2% 89 ±5% 42 ±3% 8 ±1%
t = 90 108 ±5% 106 ±7% 80 ±6% 28 ±3% 5 ±4%
t = 15 103 ±2% 103 ±3% 88 ±4% 79 ±7% –
t = 30 100 ±2% 104 ±4% 83 ±3% 45 ±4% –
t = 60 103 ±2% 104 ±5% 71 ±3% 14 ±2% –
t = 90 105 ±2% 105 ±7% 73 ±5% 11 ±4% –
Bold numbers are significant compared to the control.
significant effect on fEPSP amplitudes, either in slices taken from adult rats (Fig. 1b, n = 6) or from young rats (Fig. 1d, n = 8). The fEPSP amplitudes are listed in Table 1. Effects of DMAIII on fEPSP Bath application of DMAIII affected the amplitudes of the fEPSPs evoked in a concentration-dependent manner in slices taken from both adult (Figs. 2a, b) and young animals (Figs. 2c, d). The onset of the blocking effect varied with the concentration of DMAIII. Original recordings with and without application of 10 μmol/l DMAIII are shown in Fig. 2a (adult rats) and Fig. 2c (young rats). In adult rats, application of 1 μmol/l DMAIII (Fig. 2b, n = 5) produced a slight decrease in the fEPSP amplitudes to about 80% of the control amplitudes, which became significant 60 min after application. Application of 10 μmol/l DMAIII (Fig. 2b, n = 6) led to a greater reduction of the fEPSP amplitudes to about 30% of the control values, which was significant at all points in time. Application of 100 μmol/l DMAIII (Fig. 2b, n = 2) resulted in a complete depression of fEPSP amplitudes, starting 30 min after bath-application of the substance. The depressant effect was not reversible after a 30min washout of the DMAIII. In slices of young rats, the depres-
sant effect of DMAIII was more pronounced than in those taken from adult ones. Application of 1 μmol/l DMAIII (Fig. 2d, n = 4) produced a decrease in the fEPSP amplitudes to about 70% of the control amplitudes, which became significant 30 min after application. Application of 10 μmol/l DMAIII (Fig. 2d, n = 4) led to a total depression of the fEPSP amplitudes to about 12% of the control values, which was not reversible after a 30 min washout of the DMAIII . LTP control measurements The potentiation of the fEPSP amplitudes after the highfrequency stimulus (HFS) was lower in control slices taken from young rats (Figs. 3d, 4d, n = 6) than in those taken from adult ones (Figs. 3b, 4b, n = 7). Mean fEPSP amplitudes are listed in Table 2 for different points in time and, immediately after the HFS, were about 185% of baseline reference in slices from adult rats and 170% in those taken from young rats. Effects of DMAV on LTP To test whether DMAV affects the induction of the NMDAdependent LTP at the Schaffer collateral-CA1 synapse,
Fig. 1. Effect of dimethylarsinic acid (DMAV) on evoked postsynaptic potentials (fEPSPs) of adult and young rats. Typical traces of postsynaptic responses (fEPSPs) in the CA1 dentritic layer after stimulation of Schaffer collaterals without and with administration of 100 μmol/l DMAV in adult (a) and young rats (c). Diagrams of mean values ± S.E.M. of the fEPSP amplitudes (normalized to the average of 10-min baseline responses) under control conditions (without DMAV, white dots) and after administration of 100 μmol/l DMAV (black triangles) in adult (b) and young (d) rats. DMAV was administered at time = 0 and washed out after 60 min. Arrows indicate the points in time that were statistically analyzed, asterisks indicate a significant difference between the values obtained and the control values.
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Fig. 2. Effect of dimethylarsinous acid (DMAIII) on evoked postsynaptic potentials (fEPSPs) in adult and young rats. Typical traces of postsynaptic responses (fEPSPs) in the CA1 dentritic layer after stimulation of Schaffer collaterals without and with administration of 10 μmol/l DMAIII in adult (a) and young rats (c). Diagrams of mean values ± S.E.M. of the fEPSP amplitudes (normalized to the average of 10 min baseline responses) under control conditions (without DMAIII, white dots) and after administration of 1 μmol/l (light-grey diamonds), 10 μmol/l (dark-grey diamonds) and 100 μmol/l DMAIII (black diamonds) in adult (b) and young (d) rats. DMAIII was administered at time = 0 and washed out after 60 min. Arrows indicate the points in time that were statistically analyzed, asterisks indicate a significant difference between the values obtained and the control values.
100 μmol/l DMAV was applied for 30 min, and the LTP stimulus was applied 15 min before washout. Original recordings are shown in Figs. 3a, c. Application of 100 μmol/l DMAV did not affect the fEPSP amplitudes significantly either in slices taken from adult rats (Fig. 3b, n = 6) or in those taken from young rats (Fig. 3d, n = 11).
Effects of DMAIII on LTP Original recordings with and without application of DMAIII are shown in Fig. 4a (adult rats) and Fig. 4c (young rats). Overall, the magnitude of the fEPSP enhancement was found to be reduced in slices treated with DMAIII compared to the values
Fig. 3. Effect of dimethylarsinic acid (DMAV) on long-term potentiation (LTP) in adult and young rats. Typical traces of postsynaptic responses (fEPSPs) in the CA1 dentritic layer before (pre) and after (post) high-frequency stimulation of the Schaffer collaterals; original recordings were made without and with administration of 100 μmol/l DMAV recorded 60 min after high-frequency stimulation in adult (a) and young (c) rats. Diagrams of mean values±S.E.M. of the fEPSP amplitudes (normalized to the average of 10-min baseline responses) under control conditions (white dots) and after administration of 100 μmol/l (black triangles) DMAV in adult (b) and young (d) rats. DMAV was administered 15 min before application of the LTP stimulus (time=0) and washed out 15 min after the LTP stimulus.
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Fig. 4. Effect of dimethylarsinous acid (DMAIII) on long-term potentiation (LTP) in adult and young rats. Typical traces of postsynaptic responses (fEPSPs) in the CA1 dentritic layer before (pre) and after (post) high-frequency stimulation of the Schaffer collaterals; original recordings were made without and with administration of 10 μmol/l DMAIII recorded 60 min after high-frequency stimulation in adult (a) and young (c) rats. Diagrams of mean values ± S.E.M. of the fEPSP amplitudes (normalized to the average of 10 min baseline responses) under control conditions (white dots) and after administration of 1 μmol/l (light-grey diamonds), 10 μmol/ l (dark-grey diamonds) and 100 μmol/l DMAIII (black diamonds) in adult (b) and young (d) rats. DMAIII was administered 15 min before application of the LTP stimulus (time = 0) and washed out 15 min after the LTP stimulus. Arrows indicate the points in time that were statistically analyzed, asterisks indicate a significant difference between the values obtained and the control values.
established in the control slices. The blocking effects were reinforced with increasing concentrations of DMAIII. In adult rats, application of 1 μmol/l DMAIII caused a significant reduction to about 60–70% of the potentiated amplitudes compared to the control amplitudes, beginning immediately after the HFS stimulus, but had no effect on young rats. At a concentration of 10 μmol/l, the postsynaptic potentiation failed completely in slices taken both from adult (Fig. 4b) and from young rats (Fig. 4d), and the average fEPSP amplitudes came to about 80% (adult rats) and 66% (young rats) of the pre-HFS stimulus baseline reference. In adult rats, 25 μmol/l DMAIII led to a reduction in the amplitudes to about 40% of the baseline reference before HFS. Discussion The main finding of this paper may be that DMAIII blocks excitatory transmission at the hippocampal Schaffer collateral-
CA1 synapse in a concentration-dependent manner. The blocking effects are considerably greater in slices taken from young rats than in those from adult rats. In contrast, DMAV, even at the high concentration of 100 μmol/l, exerts no effects, either on the fEPSP or on the LTP in slices of young and adult rats. The impairment of excitatory synaptic transmission at the Schaffer collateral-CA1 synapse by DMAIII indicates that this effect is caused by the direct or indirect action of this substance on postsynaptic glutamatergic receptors. Since DMAIII considerably reduces the induction of the NMDA-dependent LTP as well as the evoked fEPSP amplitudes, it is likely that the activity of the ionotropic NMDA and AMPA (alpha-amino-3-hydroxy5-methylisoxazole-4-propionate) receptors is disturbed by this substance. This is in line with previous findings that DMAIII, applied in the same concentration range, significantly reduces membrane currents elicited by heterologously expressed NMDA receptors in Xenopus oocytes (Krüger et al., 2006a). However, no conclusion can be drawn as to whether DMAIII
Table 2 Mean fEPSP amplitudes ± SEM compared to the baseline reference before HFS LTP
Adult rats
Point in time CTRL 100 μmol/l DMAV 1 μmol/l DMAIII 10 μmol/l DMAIII 25 μmol/l DMAIII
t = 15 184 ± 6% 183 ± 5% 128 ± 9% 125 ± 4% 89 ± 3%
Young rats t = 30 186 ± 5% 181 ± 11% 121 ± 10% 94 ± 5% 56 ± 4%
Bold numbers are significant compared to the control.
t = 60 182 ± 6% 186 ± 12% 114 ± 8% 80 ± 3% 41 ± 3%
t = 90 182 ± 7% 182 ± 10% 111 ± 9% 79 ± 4% 42 ± 4%
t = 15 171 ± 4% 170 ± 8% 162 ± 8% 104 ± 9% –
t = 30 166 ± 3% 162 ± 9% 155 ± 7% 71 ± 9% –
t = 60 162 ± 4% 149 ± 11% 153 ± 7% 67 ± 8% –
t = 90 154 ± 4% 143 ± 12% 147 ± 8% 66 ± 5% –
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acts directly on the receptor channels, via indirect alteration of the receptor-surrounding membrane organization, or via intracellular structures (cf. Ficker et al., 2004). It cannot be ruled out that the disturbing effects of DMAIII on synaptic transmission are additionally influenced by changes in the neurotransmitter levels of dopamine, norepinephrine and 5hydroxytryptamine or by cytotoxic effects (Dopp et al., 2005). Furthermore, various pre-synaptic effects, for example the reduction of action potentials or the blockade of Ca2+-channels, could also be involved in the mechanisms of action of this derivative (Hsu et al., 1993; Florea et al., 2005). The developing rat brain is obviously more susceptible to impairments caused by DMAIII, since slices from young rats, as opposed to slices from adult rats, showed an increasing sensitivity to the depressing effects of this substance. This could possibly be explained by changes in the abundance and the molecular composition of glutamate receptors of the AMPA (Monyer et al., 1991) and NMDA type (Kato et al., 1991; Kirson et al., 1999; Monyer et al., 1994), especially during the first four postnatal weeks, in the hippocampus of rats. However, it is also conceivable that DMAIII accumulates to an increasing degree in hippocampal slices from young rats due to the reduced extent of myelination in these animals. In contrast to DMAIII, pentavalent DMA is completely ineffective against the evoked fEPSP and LTP in the hippocampal slice system. This is amazing, since DMAV has been reported to block both AMPA- and NMDA-dependent membrane currents in the Xenopus oocyte expression system (Krüger et al., 2006a). The absence of effects on the hippocampal synapse shows that the action of drugs, observed at the level of single identified cells, does not allow simple and certain prediction of their effects on complex tissues of the central nervous system. Comparing the effects of the tri- and pentavalent DMA species on excitatory synaptic transmission, our results are in agreement with previous investigations which demonstrated that DMAIII exerts stronger cytotoxic effects than the pentavalent species (Cohen et al., 2002; Styblo et al., 2000, Vahter, 2002). Furthermore, compared to the effects of inorganic arsenite on long-term potentiation in rat hippocampal slices, the organic arsenical DMAIII is the stronger neurotoxicant (Krüger et al., 2006b). Since arsenic compounds penetrate the blood–brain barrier and accumulate in the brain (Zheng et al., 1991; Rodriguez et al., 2001), central neurological disorders such as neuropathy, encephalopathy, confusion and disorientation occur after arsenic intoxication (Franzblau and Lilis, 1989; Ghariani et al., 1991; Luong and Nguyen, 1999). The disturbing effects of DMAIII on the glutamate receptors in the present slice preparation in principle agree with these symptoms. Furthermore, it should be noted that in our experiments the arsenicals were applied to the slices only for short time periods (30–60 min) and that the effects should therefore be classified as acute neurotoxic effects. It is conceivable that DMAIII causes more pronounced impairments when applied under more chronic conditions and that DMAV needs long-term exposure for it to have neurotoxic effects. Concerning the toxicity of inorganic arsenicals and their triand pentavalent metabolic intermediates MMA and DMA (Petrick et al., 2000; Styblo et al., 2000; Vega et al., 2001), it is
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concluded that the pentavalent and especially the trivalent methylated arsenicals may significantly contribute to the cytotoxic effects of inorganic arsenite (Styblo et al., 2000; Vahter, 2002). A comparison of the effective concentrations found in this study with those initiating cyto- and genotoxic effects in CHO cells (Dopp et al., 2004) shows that the respective concentration ranges are apparently identical. Unfortunately, there is little information on the concentrations of arsenic found in human tissues. Chowdhury et al. (2003) calculated a skin tissue level of approximately 76 μmol/l in Bangladesh residents consuming arsenic-contaminated water. In arsenic poisoning, blood arsenic content is up to 4 mg kg− 1 and brain content is about 1.9 mg kg− 1 (International Agency for Research on Cancer, 1980). Thus, the concentrations of the methylated arsenic species used in this study cover the normal range as well as the concentrations to be expected in cases of intoxication. Although arsenicals induced pathological changes in brain tissues in vivo (Fengyuan et al., 2005), data concerning the deposits and the proportion of the different arsenicals in brain tissue are currently not available. Besides the well-known cytotoxic effects of organic arsenicals, it can be assumed from this study that functional impairments of synaptic activity contribute to the neurotoxic symptoms of arsenic intoxication. The inhibitory effects of DMAIII on the excitatory synapse may contribute to a general decrease in the neuronal activity of the hippocampus as well as in the whole nervous system. They have important implications with regard to neuronal functions and may explain the impairment of processes connected with learning and memory. This is in line with recent studies (Wasserman et al., 2004; Wright et al., 2006), in which a significant correlation between children's intellectual and memory functions and AS levels is described. However, comparing the present data obtained from rat nervous tissue with human toxicological data, one has to take into consideration that rats are in general less sensitive to arsenic than humans (Odanaka et al., 1980). In summary, the effects of the trivalent dimethylated arsenical on hippocampal synapses point to the neurotoxic potential of this substance under acute and possibly chronic conditions. The fact that DMAV had no effect on synaptic transmission of this kind indicates that the trivalent compound is more toxic than the pentavental compound following acute exposures. Acknowledgments We are grateful to Prof. W. Cullen, University of British Columbia, Vancouver, Canada for kindly donating dimethyliodoarsine (Me2AsI), and to S. Sasikanthan, E. Nass and I. Winkelhues for excellent technical assistance. The project was supported by Deutsche Forschungsgemeinschaft (DFG MU 1377/3-2, MU 1377/3-3). References Anderson, W.W., Collingridge, G.L., 2001. The LTP Program: a data acquisition program for on-line analysis of long-term potentiation and other synaptic events. J. Neurosci. Methods 15, 71–83.
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Toxicology and Applied Pharmacology 225 (2007) 40 – 46 www.elsevier.com/locate/ytaap
Effects of dimethylarsinic and dimethylarsinous acid on evoked synaptic potentials in hippocampal slices of young and adult rats Katharina Krüger a,⁎,1 , Hendrik Repges a,1 , Jörg Hippler c , Louise M. Hartmann c , Alfred V. Hirner c , Heidrun Straub a , Norbert Binding b , Ulrich Mußhoff a a b
Institut für Physiologie I, Universitätsklinikum Münster, Robert-Koch-Straße 27a, D-48149 Münster, Germany Institut für Arbeitsmedizin, Universitätsklinikum Münster, Robert-Koch-Straße 51, D-48149 Münster, Germany c Institut für Umweltanalytik, Universität Duisburg-Essen, Universitätsstraße 3-5, D-45141 Essen, Germany Received 21 March 2007; revised 6 July 2007; accepted 18 July 2007 Available online 25 July 2007
Abstract In this study, the effects of pentavalent dimethylarsinic acid ((CH3)2AsO(OH); DMAV) and trivalent dimethylarsinous acid ((CH3)2As(OH); DMAIII) on synaptic transmission generated by the excitatory Schaffer collateral-CA1 synapse were tested in hippocampal slices of young (14–21 day-old) and adult (2–4 month-old) rats. Both compounds were applied in concentrations of 1 to 100 μmol/l. DMAV had no effect on the amplitudes of evoked fEPSPs or the induction of LTP recorded from the CA1 dendritic region either in adult or in young rats. However, application of DMAIII significantly reduced the amplitudes of evoked fEPSPs in a concentration-dependent manner with a total depression following application of 100 μmol/l DMAIII in adult and 10 μmol/l DMAIII in young rats. Moreover, DMAIII significantly affected the LTP-induction. Application of 10 μmol/l DMAIII resulted in a complete failure of the postsynaptic potentiation of the fEPSP amplitudes in slices taken both from adult and young rats. The depressant effect was not reversible after a 30-min washout of the DMAIII. In slices of young rats, the depressant effects of DMAIII were more pronounced than in those taken from adult ones. Compared to the (absent) effect of DMAV on synaptic transmission, the trivalent compound possesses a considerably higher neurotoxic potential. © 2007 Elsevier Inc. All rights reserved. Keywords: Hippocampus; Slice; Synaptic transmission; Organoarsenicals; DMA; Long-term potentiation
Introduction Chronic environmental and occupational exposure to arsenic compounds has been associated with numerous adverse health effects, among them lung, skin and other cancers as well as abnormal skin pigmentation, keratosis, hypertension, respiratory disease, gastrointestinal disturbances, anemia and splenomegaly (see, e.g., review, Hall, 2002). Neurological effects of arsenic after occupational or environmental exposure or accidental intoxication, such as subclinical nerve injuries (Lagerkvist and Zetterlund, 1994), delirium and encephalopathy (Morton and Caron, 1989), peripheral neuropathies (Gerr et al., 2000; Mazumder et al., 1992), ⁎ Corresponding author. Institute of Physiology I Robert-Koch-Str. 27a, D48149 Münster, Germany. Fax: +49 251 8355551. E-mail address: [email protected] (K. Krüger). 1 These authors contributed equally to this work. 0041-008X/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.taap.2007.07.007
and symptoms including loss of hearing and taste, blurred vision, tingling and numbness of the limbs, and decrease in muscle strength (Liu et al., 2002; Luong and Nguyen, 1999), have been reported. Furthermore, several investigations revealed that arsenicals have an influence on learning and memory. In children, chronic exposure to inorganic arsenic via drinking water resulted in a dose-dependent reduction in intellectual functions (Wasserman et al., 2004; Wright et al., 2006). Alterations in memory and attention have been observed in adolescents after chronic exposure to high levels of arsenic (Tsai et al., 2003). In experimental studies, behavioral alterations have been observed in rats following sodium arsenite treatment (Rodriguez et al., 2001). Effects of arsenite on brain monoamine concentrations have been found in experimental animals (Delgado et al., 2000). While the clinical outcomes of arsenic poisoning are well documented, the toxicological mechanisms involved are still not completely understood. One reason is that inorganic arsenic
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compounds (iA III and iAV ) are rapidly metabolized to methylated pentavalent and trivalent species (monomethylarsonic acid (MMAV ), monomethylarsonous acid (MMA III ), dimethylarsinic acid (DMAV ), and dimethylarsinous acid (DMAIII); Aposhian et al., 2000; Thomas et al., 2004). In most cases, it is not known whether the inorganic compounds are the ultimate toxins or their organic metabolites. Biomethylation of inorganic arsenic has been considered a major detoxification pathway for a long time. This is now questionable, since pentavalent and especially trivalent methylated arsenicals exhibit cytotoxicity (Petrick et al., 2000; Styblo et al., 2000; Cohen et al., 2002) and influence cell proliferation and cytokine secretion in human epidermal keratinocytes (Vega et al., 2001). Viability testing with these keratinocytes showed increasing toxicities of arsenicals in the following order: iAV b MMAV b DMAV b DMAIII b MMAIII b iAIII. From these experimental studies, it is concluded that methylated arsenicals, especially the trivalent species, may significantly contribute to the adverse effects of inorganic arsenite (Styblo et al., 2000; Vahter, 2002). The aim of this study was to investigate a possible contribution of the methylated arsenic metabolites DMAV and DMAIII to the known neurotoxic effects induced by exposure to inorganic arsenite. Hippocampal slices from rat brain were chosen to analyze the effects of pentavalent and trivalent DMA on synaptic transmission at identified synaptic sites. Materials and methods Substances. Dimethylarsinic acid (DMAV) was purchased from Strem (Kehl, Germany). Dimethyliodoarsine (Me2AsI) was kindly donated by Prof. W. Cullen (University of British Columbia, Vancouver, Canada). Preparation of dilute solutions of this iodide precursor results in the formation of the corresponding acid, dimethylarsinous acid (DMAIII) (Gong et al., 2001; Millar et al., 1960). Since only a small amount of DMAIII was available, the measurements with DMAIII were reduced in number. All chemicals were of analytical grade or the highest quality available. In a series of experiments on Xenopus oocytes (Krüger et al., 2006a), the stability of both arsenicals was tested by HPLC-hydride generation atomic fluorescence spectroscopy (HPLC-HG-AFS) using the method of Le et al. (2000). In these experiments, DMAIII and DMAV were added to a modified Ringer's solution which served as diluting agent and was composed of (in mmol/l): NaCl 115, KCl 2, CaCl2 1.8, HEPES 10; pH 7.2. DMAV was found to be stable, i.e., there was no detectable loss in concentration or species transformation over a minimum 2-month period. DMAIII was both volatile and highly susceptible to oxidation; within 7 days less than 20% of the original DMAIII concentration was present and by 30 days no DMAIII could be detected. Around half of the DMAIII losses were accounted for by oxidation of the DMAIII to DMAV, the remainder was presumably volatilized. Storage temperature did not significantly affect the stability of DMAIII. Stock solutions of 1 mmol/L DMAV and DMAIII were prepared in artificial cerebrospinal fluid (ACSF) and stored at 4 °C in glass bottles. The ACSF contained (in mmol/l) NaCl 124, KCl 4, CaCl2 2, NaH2PO4 1.24, NaHCO3 26, MgCl2 1.3 and glucose 10. Due to its high volatility and sensitivity to oxidation, solutions of DMAIII were always prepared immediately before each experiment and discarded if not used within 2 days. Preparation of hippocampal slices. The technique for preparation of hippocampal tissue slices has been described in detail elsewhere (Musshoff et al., 2002). Briefly, 32 adult rats (Wistar, 220–360 g, mean 290 g, 2–4 months old, male) and 25 young rats (Wistar, 25–60 g, mean 39 g, 14–21 days old, mean 17 days old) were decapitated under ether anesthesia. All procedures utilizing animals were performed in accordance with regulations through the German law and were approved by the Bezirksregierung Münster. Both hippocampi were excised from the brain and submerged in chilled (2–4 °C) ACSF for about
41
1 min. Slices of 500 μm thickness were cut from the dorsal hippocampus and pre-incubated in ACSF in a submersion chamber for at least 60 min at 28 °C. The ACSF was constantly bubbled with 95% O2 and 5% CO2. For electrophysiological investigations, slices were maintained in a submergedtype recording chamber with the temperature (32 °C), pH (7.4) and flow rate (4 ml/min, bath volume 1 ml) of the ACSF being continuously monitored. Stock solutions of DMAV or DMAIII were diluted immediately prior to experiments and added to the bath solution in concentrations of 1, 10, 25 or 100 μmol/l. Induction of excitatory post-synaptic potentials. fEPSPs were elicited by Schaffer collateral stimulation through a bipolar stimulation electrode and recorded as extracellular field potentials with ACSF-filled glass recording electrodes (0.5–1.5 MΩ) placed in the stratum radiatum of the CA1 region. The synaptic response to a standard test stimulus (0.033 Hz) was monitored until a stable recording was obtained, and the input–output relationship was then determined. The stimulus strength (0.2–2.5 mA) producing a response of approximately 50% of the maximal response amplitude was determined and used for all subsequent experiments. Only synaptic potentials with more than 0.2 mV and without superimposed population spikes were used for the experiments. After stable baseline recording of the responses for 20 min, DMAV or DMAIII was bath-applied to the hippocampal slices for 60 min and then washed out for a further 30 min. The evoked synaptic responses were recorded every 30 s during bathapplication of DMAV or DMAIII and for a further 30 min after washout. Induction of long-term potentiation (LTP). The baseline presynaptic stimulation was delivered at 0.033 Hz for 20 min using a stimulation intensity evoking approximately 50% of the maximal post-synaptic responses. Following the application of a high-frequency stimulus (HFS: 100 Hz, 1 s duration), stimulation was again delivered at 0.033 Hz but now resulted in a potentiated synaptic response which was recorded for a further 90 min. DMAV or DMAIII was bath-applied to the hippocampal slices 15 min before LTP induction and washed out 15 min after setting the high-frequency stimulus. Analyses. The evoked synaptic responses were recorded and analyzed with a personal computer using custom-developed software (LTP program, version 2.30D; Anderson and Collingridge, 2001) and a digital oscilloscope. The fEPSPs were quantified by measurements of the amplitude of the synaptic responses. Each of the amplitudes of the fEPSPs obtained during DMA application was normalized to the average amplitude of the 10 min baseline recordings of the fEPSPs acquired before DMAV or DMAIII application. For LTP experiments, the amplitudes of the post-tetanic responses were normalized to the average of 10-min baseline responses obtained before setting the LTP stimulus. The significance of the differences between the means was calculated for different points in time (t = 15, 30, 60, 90 min), using a t-test or Mann–Whitney rank sum test. Values were considered significantly different if p ≤ 0.05. In the text, values are shown as mean ± S.E.M.
Results fEPSP control measurements To test whether the organoarsenic compounds affect excitatory transmission at the Schaffer collateral-CA1 synapse, evoked fEPSPs were measured under control conditions (ACSF) and after administration of the organoarsenic compounds in ACSF. Under control conditions, the amplitudes of the fEPSPs remained stable compared to the baseline reference over the whole range of 90 min. The mean fEPSP amplitudes and SEM are listed in Table 1 for different points in time and, 15 min after the HFS, were 100% in adult rats (Figs. 1b, 2b, n=6) and 103% in young rats (Figs. 1d, 2d, n=7). Effects of DMAV on fEPSP Original recordings with and without bath application of 100 μmol/l DMAV are shown in Fig. 1a for adult and Fig. 1c for young rats. Bath application of 100 μmol/l DMAV had no
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Table 1 Mean fEPSP amplitudes ± SEM compared to the baseline reference fEPSP
Adult rats
Point in time CTRL 100 μmol/l DMAV 1 μmol/l DMAIII 10 μmol/l DMAIII 100 μmol/l DMAIII
t = 15 100 ±3% 96 ±3% 98 ±2% 90 ±1% 45 ±2%
Young rats t = 30 100 ±3% 96 ±3% 91 ±4% 72 ±3% 9 ±1%
t = 60 106 ±3% 101 ±2% 89 ±5% 42 ±3% 8 ±1%
t = 90 108 ±5% 106 ±7% 80 ±6% 28 ±3% 5 ±4%
t = 15 103 ±2% 103 ±3% 88 ±4% 79 ±7% –
t = 30 100 ±2% 104 ±4% 83 ±3% 45 ±4% –
t = 60 103 ±2% 104 ±5% 71 ±3% 14 ±2% –
t = 90 105 ±2% 105 ±7% 73 ±5% 11 ±4% –
Bold numbers are significant compared to the control.
significant effect on fEPSP amplitudes, either in slices taken from adult rats (Fig. 1b, n = 6) or from young rats (Fig. 1d, n = 8). The fEPSP amplitudes are listed in Table 1. Effects of DMAIII on fEPSP Bath application of DMAIII affected the amplitudes of the fEPSPs evoked in a concentration-dependent manner in slices taken from both adult (Figs. 2a, b) and young animals (Figs. 2c, d). The onset of the blocking effect varied with the concentration of DMAIII. Original recordings with and without application of 10 μmol/l DMAIII are shown in Fig. 2a (adult rats) and Fig. 2c (young rats). In adult rats, application of 1 μmol/l DMAIII (Fig. 2b, n = 5) produced a slight decrease in the fEPSP amplitudes to about 80% of the control amplitudes, which became significant 60 min after application. Application of 10 μmol/l DMAIII (Fig. 2b, n = 6) led to a greater reduction of the fEPSP amplitudes to about 30% of the control values, which was significant at all points in time. Application of 100 μmol/l DMAIII (Fig. 2b, n = 2) resulted in a complete depression of fEPSP amplitudes, starting 30 min after bath-application of the substance. The depressant effect was not reversible after a 30min washout of the DMAIII. In slices of young rats, the depres-
sant effect of DMAIII was more pronounced than in those taken from adult ones. Application of 1 μmol/l DMAIII (Fig. 2d, n = 4) produced a decrease in the fEPSP amplitudes to about 70% of the control amplitudes, which became significant 30 min after application. Application of 10 μmol/l DMAIII (Fig. 2d, n = 4) led to a total depression of the fEPSP amplitudes to about 12% of the control values, which was not reversible after a 30 min washout of the DMAIII . LTP control measurements The potentiation of the fEPSP amplitudes after the highfrequency stimulus (HFS) was lower in control slices taken from young rats (Figs. 3d, 4d, n = 6) than in those taken from adult ones (Figs. 3b, 4b, n = 7). Mean fEPSP amplitudes are listed in Table 2 for different points in time and, immediately after the HFS, were about 185% of baseline reference in slices from adult rats and 170% in those taken from young rats. Effects of DMAV on LTP To test whether DMAV affects the induction of the NMDAdependent LTP at the Schaffer collateral-CA1 synapse,
Fig. 1. Effect of dimethylarsinic acid (DMAV) on evoked postsynaptic potentials (fEPSPs) of adult and young rats. Typical traces of postsynaptic responses (fEPSPs) in the CA1 dentritic layer after stimulation of Schaffer collaterals without and with administration of 100 μmol/l DMAV in adult (a) and young rats (c). Diagrams of mean values ± S.E.M. of the fEPSP amplitudes (normalized to the average of 10-min baseline responses) under control conditions (without DMAV, white dots) and after administration of 100 μmol/l DMAV (black triangles) in adult (b) and young (d) rats. DMAV was administered at time = 0 and washed out after 60 min. Arrows indicate the points in time that were statistically analyzed, asterisks indicate a significant difference between the values obtained and the control values.
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Fig. 2. Effect of dimethylarsinous acid (DMAIII) on evoked postsynaptic potentials (fEPSPs) in adult and young rats. Typical traces of postsynaptic responses (fEPSPs) in the CA1 dentritic layer after stimulation of Schaffer collaterals without and with administration of 10 μmol/l DMAIII in adult (a) and young rats (c). Diagrams of mean values ± S.E.M. of the fEPSP amplitudes (normalized to the average of 10 min baseline responses) under control conditions (without DMAIII, white dots) and after administration of 1 μmol/l (light-grey diamonds), 10 μmol/l (dark-grey diamonds) and 100 μmol/l DMAIII (black diamonds) in adult (b) and young (d) rats. DMAIII was administered at time = 0 and washed out after 60 min. Arrows indicate the points in time that were statistically analyzed, asterisks indicate a significant difference between the values obtained and the control values.
100 μmol/l DMAV was applied for 30 min, and the LTP stimulus was applied 15 min before washout. Original recordings are shown in Figs. 3a, c. Application of 100 μmol/l DMAV did not affect the fEPSP amplitudes significantly either in slices taken from adult rats (Fig. 3b, n = 6) or in those taken from young rats (Fig. 3d, n = 11).
Effects of DMAIII on LTP Original recordings with and without application of DMAIII are shown in Fig. 4a (adult rats) and Fig. 4c (young rats). Overall, the magnitude of the fEPSP enhancement was found to be reduced in slices treated with DMAIII compared to the values
Fig. 3. Effect of dimethylarsinic acid (DMAV) on long-term potentiation (LTP) in adult and young rats. Typical traces of postsynaptic responses (fEPSPs) in the CA1 dentritic layer before (pre) and after (post) high-frequency stimulation of the Schaffer collaterals; original recordings were made without and with administration of 100 μmol/l DMAV recorded 60 min after high-frequency stimulation in adult (a) and young (c) rats. Diagrams of mean values±S.E.M. of the fEPSP amplitudes (normalized to the average of 10-min baseline responses) under control conditions (white dots) and after administration of 100 μmol/l (black triangles) DMAV in adult (b) and young (d) rats. DMAV was administered 15 min before application of the LTP stimulus (time=0) and washed out 15 min after the LTP stimulus.
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Fig. 4. Effect of dimethylarsinous acid (DMAIII) on long-term potentiation (LTP) in adult and young rats. Typical traces of postsynaptic responses (fEPSPs) in the CA1 dentritic layer before (pre) and after (post) high-frequency stimulation of the Schaffer collaterals; original recordings were made without and with administration of 10 μmol/l DMAIII recorded 60 min after high-frequency stimulation in adult (a) and young (c) rats. Diagrams of mean values ± S.E.M. of the fEPSP amplitudes (normalized to the average of 10 min baseline responses) under control conditions (white dots) and after administration of 1 μmol/l (light-grey diamonds), 10 μmol/ l (dark-grey diamonds) and 100 μmol/l DMAIII (black diamonds) in adult (b) and young (d) rats. DMAIII was administered 15 min before application of the LTP stimulus (time = 0) and washed out 15 min after the LTP stimulus. Arrows indicate the points in time that were statistically analyzed, asterisks indicate a significant difference between the values obtained and the control values.
established in the control slices. The blocking effects were reinforced with increasing concentrations of DMAIII. In adult rats, application of 1 μmol/l DMAIII caused a significant reduction to about 60–70% of the potentiated amplitudes compared to the control amplitudes, beginning immediately after the HFS stimulus, but had no effect on young rats. At a concentration of 10 μmol/l, the postsynaptic potentiation failed completely in slices taken both from adult (Fig. 4b) and from young rats (Fig. 4d), and the average fEPSP amplitudes came to about 80% (adult rats) and 66% (young rats) of the pre-HFS stimulus baseline reference. In adult rats, 25 μmol/l DMAIII led to a reduction in the amplitudes to about 40% of the baseline reference before HFS. Discussion The main finding of this paper may be that DMAIII blocks excitatory transmission at the hippocampal Schaffer collateral-
CA1 synapse in a concentration-dependent manner. The blocking effects are considerably greater in slices taken from young rats than in those from adult rats. In contrast, DMAV, even at the high concentration of 100 μmol/l, exerts no effects, either on the fEPSP or on the LTP in slices of young and adult rats. The impairment of excitatory synaptic transmission at the Schaffer collateral-CA1 synapse by DMAIII indicates that this effect is caused by the direct or indirect action of this substance on postsynaptic glutamatergic receptors. Since DMAIII considerably reduces the induction of the NMDA-dependent LTP as well as the evoked fEPSP amplitudes, it is likely that the activity of the ionotropic NMDA and AMPA (alpha-amino-3-hydroxy5-methylisoxazole-4-propionate) receptors is disturbed by this substance. This is in line with previous findings that DMAIII, applied in the same concentration range, significantly reduces membrane currents elicited by heterologously expressed NMDA receptors in Xenopus oocytes (Krüger et al., 2006a). However, no conclusion can be drawn as to whether DMAIII
Table 2 Mean fEPSP amplitudes ± SEM compared to the baseline reference before HFS LTP
Adult rats
Point in time CTRL 100 μmol/l DMAV 1 μmol/l DMAIII 10 μmol/l DMAIII 25 μmol/l DMAIII
t = 15 184 ± 6% 183 ± 5% 128 ± 9% 125 ± 4% 89 ± 3%
Young rats t = 30 186 ± 5% 181 ± 11% 121 ± 10% 94 ± 5% 56 ± 4%
Bold numbers are significant compared to the control.
t = 60 182 ± 6% 186 ± 12% 114 ± 8% 80 ± 3% 41 ± 3%
t = 90 182 ± 7% 182 ± 10% 111 ± 9% 79 ± 4% 42 ± 4%
t = 15 171 ± 4% 170 ± 8% 162 ± 8% 104 ± 9% –
t = 30 166 ± 3% 162 ± 9% 155 ± 7% 71 ± 9% –
t = 60 162 ± 4% 149 ± 11% 153 ± 7% 67 ± 8% –
t = 90 154 ± 4% 143 ± 12% 147 ± 8% 66 ± 5% –
K. Krüger et al. / Toxicology and Applied Pharmacology 225 (2007) 40–46
acts directly on the receptor channels, via indirect alteration of the receptor-surrounding membrane organization, or via intracellular structures (cf. Ficker et al., 2004). It cannot be ruled out that the disturbing effects of DMAIII on synaptic transmission are additionally influenced by changes in the neurotransmitter levels of dopamine, norepinephrine and 5hydroxytryptamine or by cytotoxic effects (Dopp et al., 2005). Furthermore, various pre-synaptic effects, for example the reduction of action potentials or the blockade of Ca2+-channels, could also be involved in the mechanisms of action of this derivative (Hsu et al., 1993; Florea et al., 2005). The developing rat brain is obviously more susceptible to impairments caused by DMAIII, since slices from young rats, as opposed to slices from adult rats, showed an increasing sensitivity to the depressing effects of this substance. This could possibly be explained by changes in the abundance and the molecular composition of glutamate receptors of the AMPA (Monyer et al., 1991) and NMDA type (Kato et al., 1991; Kirson et al., 1999; Monyer et al., 1994), especially during the first four postnatal weeks, in the hippocampus of rats. However, it is also conceivable that DMAIII accumulates to an increasing degree in hippocampal slices from young rats due to the reduced extent of myelination in these animals. In contrast to DMAIII, pentavalent DMA is completely ineffective against the evoked fEPSP and LTP in the hippocampal slice system. This is amazing, since DMAV has been reported to block both AMPA- and NMDA-dependent membrane currents in the Xenopus oocyte expression system (Krüger et al., 2006a). The absence of effects on the hippocampal synapse shows that the action of drugs, observed at the level of single identified cells, does not allow simple and certain prediction of their effects on complex tissues of the central nervous system. Comparing the effects of the tri- and pentavalent DMA species on excitatory synaptic transmission, our results are in agreement with previous investigations which demonstrated that DMAIII exerts stronger cytotoxic effects than the pentavalent species (Cohen et al., 2002; Styblo et al., 2000, Vahter, 2002). Furthermore, compared to the effects of inorganic arsenite on long-term potentiation in rat hippocampal slices, the organic arsenical DMAIII is the stronger neurotoxicant (Krüger et al., 2006b). Since arsenic compounds penetrate the blood–brain barrier and accumulate in the brain (Zheng et al., 1991; Rodriguez et al., 2001), central neurological disorders such as neuropathy, encephalopathy, confusion and disorientation occur after arsenic intoxication (Franzblau and Lilis, 1989; Ghariani et al., 1991; Luong and Nguyen, 1999). The disturbing effects of DMAIII on the glutamate receptors in the present slice preparation in principle agree with these symptoms. Furthermore, it should be noted that in our experiments the arsenicals were applied to the slices only for short time periods (30–60 min) and that the effects should therefore be classified as acute neurotoxic effects. It is conceivable that DMAIII causes more pronounced impairments when applied under more chronic conditions and that DMAV needs long-term exposure for it to have neurotoxic effects. Concerning the toxicity of inorganic arsenicals and their triand pentavalent metabolic intermediates MMA and DMA (Petrick et al., 2000; Styblo et al., 2000; Vega et al., 2001), it is
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concluded that the pentavalent and especially the trivalent methylated arsenicals may significantly contribute to the cytotoxic effects of inorganic arsenite (Styblo et al., 2000; Vahter, 2002). A comparison of the effective concentrations found in this study with those initiating cyto- and genotoxic effects in CHO cells (Dopp et al., 2004) shows that the respective concentration ranges are apparently identical. Unfortunately, there is little information on the concentrations of arsenic found in human tissues. Chowdhury et al. (2003) calculated a skin tissue level of approximately 76 μmol/l in Bangladesh residents consuming arsenic-contaminated water. In arsenic poisoning, blood arsenic content is up to 4 mg kg− 1 and brain content is about 1.9 mg kg− 1 (International Agency for Research on Cancer, 1980). Thus, the concentrations of the methylated arsenic species used in this study cover the normal range as well as the concentrations to be expected in cases of intoxication. Although arsenicals induced pathological changes in brain tissues in vivo (Fengyuan et al., 2005), data concerning the deposits and the proportion of the different arsenicals in brain tissue are currently not available. Besides the well-known cytotoxic effects of organic arsenicals, it can be assumed from this study that functional impairments of synaptic activity contribute to the neurotoxic symptoms of arsenic intoxication. The inhibitory effects of DMAIII on the excitatory synapse may contribute to a general decrease in the neuronal activity of the hippocampus as well as in the whole nervous system. They have important implications with regard to neuronal functions and may explain the impairment of processes connected with learning and memory. This is in line with recent studies (Wasserman et al., 2004; Wright et al., 2006), in which a significant correlation between children's intellectual and memory functions and AS levels is described. However, comparing the present data obtained from rat nervous tissue with human toxicological data, one has to take into consideration that rats are in general less sensitive to arsenic than humans (Odanaka et al., 1980). In summary, the effects of the trivalent dimethylated arsenical on hippocampal synapses point to the neurotoxic potential of this substance under acute and possibly chronic conditions. The fact that DMAV had no effect on synaptic transmission of this kind indicates that the trivalent compound is more toxic than the pentavental compound following acute exposures. Acknowledgments We are grateful to Prof. W. Cullen, University of British Columbia, Vancouver, Canada for kindly donating dimethyliodoarsine (Me2AsI), and to S. Sasikanthan, E. Nass and I. Winkelhues for excellent technical assistance. The project was supported by Deutsche Forschungsgemeinschaft (DFG MU 1377/3-2, MU 1377/3-3). References Anderson, W.W., Collingridge, G.L., 2001. The LTP Program: a data acquisition program for on-line analysis of long-term potentiation and other synaptic events. J. Neurosci. Methods 15, 71–83.
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