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Toluene increases acute thermonociception in mice
Behavioural Brain Research 120 (2001) 213– 220 www.elsevier.com/locate/bbr
Toluene increases acute thermonociception in mice Silvia Lorenia Cruz a,*, Nayeli Pa´ez-Martı´nez a, Francisco Pellicer b, Luis Antonio Salazar a, Carolina Lo´pez-Rubalcava a a
Departmento de Farmacobiologı´a, Cin6esta6 IPN, Apartado Postal 22026, 14000 Mexico D.F., Mexico b Di6isio´n de Neurociencias, Instituto Nacional de Psiquiatrı´a, Me´xico D.F., Mexico
Received 16 December 1999; received in revised form 02 November 2000; accepted 14 November 2000
Abstract Toluene is an abused solvent widely used in several commercial products. Recent evidence indicates that this solvent is a non-competitive inhibitor of NMDA receptors. Since NMDA receptors have been implicated in pain, this paper describes studies of the effects of increasing concentrations of inhaled toluene on nociception. Swiss Webster mice were exposed to toluene (500–8000 ppm) in static exposure chambers for 30 min. After completing the exposure period, animals were tested for nociception using the hot plate test. Toluene dose-dependently increased nociception as reflected by shorter latencies for the reflex, paw-lick and escape responses in toluene-treated mice with respect to their controls (animals exposed to air). In order to determine the possible role of opioids in this response, morphine (1 – 10 mg/kg) was injected before toluene inhalation. Toluene was not able to block morphine-induced antinocieption, however, it produced a shift of the morphine dose– response curve to lower effects, suggesting a physiological antagonism. No potentiation was seen when toluene was administered in combination with naloxone. Present results suggest that toluene increases nociception via neurotransmitter systems others than the glutamatergic. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Toluene; Nociception; Opioids; Hot-plate test; Morphine; Naloxone; Mice
1. Introduction Toluene is an industrial solvent present in many commercial products that is inhaled as a drug of abuse. Humans come into contact with it through occupational exposure or via its intentional inhalation for recreational purposes [18,27]. The mechanism of action of toluene is not completely understood, however, sound behavioral data indicate that this compound shares several effects with other central nervous system (CNS) depressants, including ethanol [21]. In agreement with these behavioral findings, recent evidence indicates that toluene, like other CNS depressants, affects ligandgated ion channel activity. Thus, toluene, at micromolar concentrations, acts as a reversible and potent non-competitive inhibitor of N-methyl-D-aspartic acid (NMDA) receptors, has no action on non-NMDA glutamate receptors [15], and inhibits nicotine receptors * Corresponding author. Tel.: +52-5-4832853; fax: +52-54832863. E-mail address: [email protected] (S.L. Cruz).
[5]. The experimental evidence on the effects of toluene on other ligand-gated channels is scarce and, therefore, less conclusive. According to Beckstead and co-workers, toluene at 0.5 mM enhances GABAA and glycine receptor activity [8]. Other groups, however, have not found changes in brain and cerebellum GABA contents in animals exposed to high concentrations of toluene [30]. As to the effects of toluene on 5HT3 receptors, preliminary data suggest that agonist-induced currents in recombinant 5HT3 receptors are enhanced in the presence of toluene [9]. The new evidence on the cellular actions of abused solvents, although limited, offers new insights for the investigation of the behavioral effects of these substances. For example, if toluene inhibits the glutamate NMDA receptor subtype, it would be interesting to examine whether its inhalation modifies pain perception. It is well established that excitatory amino acids play a significant role in nociceptive processes. Glutamate is an important neurotransmitter in primary sensory noci-
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ceptive neurons and is released in response to noxious stimuli [7,17,45]. Intrathecal injections of NMDA- and non-NMDA receptor agonists have pronociceptive effects [1,48]. On the other hand, both competitive and non-competitive NMDA receptor antagonists are effective in producing antinociception in models of persistent nociception [14,47] and, according to recent reports, also in acute models of pain [23]. In addition, a close relationship between glutamatergic and opioidergic systems exists. Opiates have been used for centuries for the treatment of pain. Opioid receptors are distributed throughout the CNS structures that are involved in transmission, modulation and sensation of pain [26,37]. The interaction between glutamate and opioids is complex. Pharmacological activation of the m receptor suppresses the magnitude of glutamate evoked postsynaptic currents through the presynaptic inhibition of the excitatory transmitter release [25]. NMDA antagonists enhance opioid antinociception [44], prevent the development of tolerance to morphine-induced analgesia and attenuate morphine withdrawal in several experimental models [10,31,32,39,43]. Thus, the relationship between NMDA and opioid receptors depends on the time of opioid exposure, previous exposure to opioid agonists, the responses under study and the relative doses of the compounds used. Based on these findings, the purpose of the present investigation was to study the acute effects of toluene on nociception in the hot plate test. The first objective was to establish a dose-response relationship for toluene effects on nociception. The second goal was to determine if an interaction between toluene and the opioidergic system existed under the circumstances of this study.
2. Material and methods
2.1. Animals Male Swiss –Webster mice (20 – 30 g) bred in our animal facilities were used in this study. Mice were housed 10 to a cage and had free access to food and water throughout the experiments. Colonies and experimental rooms were maintained under controlled light – dark cycle conditions (12:12 h, lights on at 07:00 h) with a temperature of 22 – 24°C. Testing was routinely performed between 09:00 and 15:00 h and independent groups of mice were used for each experimental condition. All procedures were performed in accordance to the regulations of the Ethics Committee of the International Association for the Study of Pain [50], and were approved by our institutional ethics committee.
2.2. Drugs Toluene 99.8% HPLC degree and naloxone hydrochloride were purchased from Sigma Chemicals Co. (St. Louis, MO). Morphine hydrochloride was obtained from Merck (Darmstadt, Germany) via the Mexican Ministry of Health. Opioids were dissolved in saline solution and injected i.p. in a volume of 0.1 ml/10 g.
2.3. Inhalation exposures Mice were exposed to air or toluene in a 29-liter static exposure chamber. In this closed system a known amount of toluene was completely volatilized to generate a concentration of 500, 1000, 2000, 4000, 6000 or 8000 ppm. The exposure chamber consisted of a chromatographic cylindrical jar covered with an acrylic lid that had injection ports and a fan that projected into the chamber above a stainless steel mesh platform [34,46]. For toluene exposure, mice were placed in the bottom of the chamber, the lid was replaced and a pre-determined amount of toluene was injected onto a filter paper located on the wire mesh. The fan was turned on within 5 s of the solvent injection to volatilize the solvent and to produce the desired concentration. Nominal vapor concentrations were confirmed with a single-wave length infrared spectrometer (Miran 1A Foxboro Analytical).
2.4. Adaptation sessions In order to diminish the influence of stress, all mice were allowed to acclimate on two consecutive days by placing them in the exposure chambers (fans functioning) for 30 min. On the third day, animals were exposed to toluene or air for 30 min and tested immediately after for nociception or motor coordination.
2.5. Hot plate test Each mouse was introduced into a glass cylinder (20 cm diameter and 25 cm height) placed at the center of a metal plate adjusted at 539 0.5°C. Within several seconds, the animal displayed three specific responses evoked by the thermal stimulus: the flexor antialgesic reflex, paw lick and escape behaviors. We considered the escape behavior as the simultaneous withdrawal of the rear limbs. The latencies to the appearance of all responses were determined and the test was concluded after 35 s to prevent tissue damage. In this model, antinociception is seen as increased latencies to the responses evaluated, while increased nociception is manifested by shorter latencies [47]. Hot plate sessions were videotaped for later measurement by an observer blind to the treatment.
S.L. Cruz et al. / Beha6ioural Brain Research 120 (2001) 213–220
2.6. Motor coordination test The effects of toluene on motor coordination were assessed using the rota-rod test. Animals were placed on a 7 cm cylinder rotating at 11 rpm and the number of falls during a 5 min session registered. Previously, the animals were trained for three consecutive trials on the rotating cylinder. For the experimental trial, animals were exposed to either air or toluene and after removal from the exposure chamber were immediately tested again on the rota-rod. The performance of each animal after air or toluene was compared to its own performance during the previous (third) training session.
2.7. Experiment I: effects of toluene on thermonociception Independent groups of animals were exposed to air or toluene (500 –8000 ppm; n =10 each) for 30 min and tested immediately after (within 5 min) in the hot plate.
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and the other was injected with saline and exposed to air. A second experimental series was established to determine the putative interaction between toluene and the opioid competitive antagonist naloxone. As in the previous experiment, control animals were injected with saline and exposed to air. The effects of low doses of naloxone (0.1 and 0.2) alone and in combination with either toluene 500 or 1000 ppm were studied on the hot plate.
2.9. Data analysis Data were analyzed with a Kruskal –Wallis one-way Analysis of Variance (ANOVA). If significant effects were observed, a Mann –Whitney U-test was used to determine differences between groups. Comparisons of the performance on the rota-rod during the last training session and after drug treatment were done by the Wilcoxon t-test. Statistical procedures were done using the Sigma Stat program (version 2.03, Jandel Scientific).
2.8. Experiment II. interaction of toluene with opiates In order to test if the effects of toluene on nociception were due to an interaction with the endogenous opioid system, the effects of toluene exposure in morphine-treated animals were studied. To this purpose, morphine (1, 3, 10 and 17 mg/kg i.p.; n = 10 each) was injected immediately before exposing the animals either to air or to a fixed concentration of toluene (4000 ppm). Two independent groups served as controls: one was injected with saline and exposed to toluene (4000 ppm)
3. Results
3.1. Experiment I: effects of toluene on nociception A 30 min exposure to toluene induced a concentration-dependent increase in nociception evaluated in the hot plate test (Fig. 1). This pronociceptive effect was manifested as shorter latencies to the appearance of the antialgesic flexor reflex, paw lick and escape responses
Fig. 1. Effects of toluene on nociception evaluated by the latencies to the appearance of the antialgesic flexor reflex, the paw-lick and the escape behaviors in mice. Independent groups of animals (n= 10, each) were exposed for 30 min to different toluene concentrations and immediately after, tested in the hot plate. Control animals (C) were exposed to air. Each point represents the mean value 9 S.E.M. *P B 0.05; Mann–Whitney U-test.
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Fig. 2. Effects of morphine (1, 3 and 10 mg/kg, i.p) alone (empty circles) and in combination with toluene (4000 ppm, filled circles) in the hot plate test. Control groups were animals injected with saline and exposed to air (S) or to 4000 ppm toluene (T). Each point represents the mean value9S.E.M. of 10 animals. *PB 0.05; Mann–Whitney U-test.
in toluene-treated animals as compared to their controls. Although no asymptotic effects were seen at the concentration range tested, the effects of 8000 ppm toluene could be considered as maximal, since it is technically impossible to observe a flexor reflex latency shorter than 1 s. It is worth mentioning that no jumps were seen during the observation period; in our experiments, the escape response consisted of the simultaneous withdrawal of both hindlimbs. As a complement of this experiment, evaluation of the effects of toluene on motor coordination revealed no significant effects at any of the concentrations tested.
In an attempt to determine if an interaction between naloxone and toluene occurred, two experiments were done. Subeffective doses/concentrations of naloxone/ toluene were tested alone and in combination in the hot plate test. When 0.1 mg/kg naloxone and 500 ppm toluene were used (Fig. 3, upper panel), no differences were found between control, toluene-treated, naloxonetreated and combined groups. Similarly, neither 1000 ppm toluene, nor 0.2 mg/kg naloxone had significant effects on any of the responses evaluated. In this case, the combination naloxone –toluene resulted in a significant decrease only in the escape response (Fig. 3, lower panel).
3.2. Experiment II: interaction of toluene with opiates Fig. 2 shows the effects of three doses of morphine (1, 3 and 10 mg/kg) alone (empty circles) and in combination with 4000 ppm toluene (filled circles) in the hot plate test. As seen in the previous experiment, toluene, at 4000 ppm, significantly decreased the latencies to all the responses evaluated as compared with those seen in control animals injected with saline and exposed to air. As expected, morphine produced antinociceptive effects in a dose-dependent fashion observed as increased latencies to the antialgesic flexor reflex, the paw lick and escape behaviors. The combined treatment of toluene with morphine also resulted in a dose-dependent increase in all the latencies to the responses evaluated, but with values that were consistently lower than those seen in animals treated with morphine alone. A higher concentration of morphine (17 mg/kg) alone, and in combination with toluene resulted in a clear analgesia in some animals that was reflected in latencies higher than the established cutoff time (35 s) making comparisons between groups impossible to establish.
4. Discussion Toluene is used in many industrial processes [3] and is voluntarily inhaled for intoxicating purposes around the world [27]. The widespread use of this substance does not correspond to our knowledge of its mechanism of action [6]. Lately, efforts have been made to study the cellular effects of abused solvents resulting in the finding that toluene affects ligand-gated ion channels. In particular, the evidence that toluene acts as a non-competitive NMDA antagonist [15] along with the documentation that NMDA antagonists possess analgesic activity, raised the question as whether or not toluene modifies nociception. We chose the hot-plate test as an experimental tool to address this issue because it allows the study of a spinally mediated response (the antialgesic flexor reflex) and two integrated behaviors (paw lick and escape) [42]. Since it is well characterized that stress interferes with nociception tests [22], in the design of the experiments, care was
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taken to minimize the influence of animal manipulation by allowing them to adapt to the exposure chamber on two consecutive days prior to the experiment. Under these conditions, toluene induced a concentration-dependent increase in nociception in the three responses evaluated. Since, as mentioned earlier, these responses are integrated at different levels of the CNS, the pronociceptive effects of toluene appear to be a general phenomenon that involves spinal and supraspinal structures. Along with the nociception experiments, we evaluated the putative disruptive effects of toluene on motor coordination in the rota-rod test, assuming that animals who were able to walk in this test would also be able to display the integrated behaviors (paw lick and escape). No significant motor impairment was seen after any of the toluene concentrations tested. These results, however, differ from those reported by Tegeris and Balster [40] who found motor impairment in mice exposed to similar toluene concentrations. These discrepancies could probably be due to differences in sensitivity in the
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models used to evaluate motor coordination. In addition, other authors [12] have pointed out that toluene, at the concentrations used in this work, increases locomotor activity. We also observed increased activity during exposure, but it did not seem to interfere with the display of the responses evaluated in the hot plate test, since all animals were able to react to the noxious stimulus more rapidly than control mice exposed to air. The mechanisms of action of abused solvents were not studied at the molecular level until recently. A few years ago, it was generally accepted that toluene effects were due to non-specific cellular changes by virtue of its actions on membrane solubility [19,28,29]. Recent investigation has focused on more specific molecular targets finding that toluene, at relevant concentrations for behavioral effects not compromising the integrity of cellular membranes, inhibits NMDA but not AMPA and kainate receptors in vitro [15]. As previously mentioned, it has been reported that competitive and noncompetitive NMDA antagonists have antinociceptive effects in several experimental paradigms [13,20,36].
Fig. 3. Upper panel: Effects of 500 ppm toluene, 0.1 mg/kg naloxone and their combination on the antialgesic flexor reflex, the paw lick and the escape behaviors. Lower panel: Effects of 1000 ppm toluene and 0.2 mg/kg naloxone alone and in combination on the three responses evaluated in the hot plate test. Control animals (white bars) were injected with saline and exposed to air. Each bar represents the mean value 9S.E.M. of 10 mice. *P B0.05; Mann–Whitnet U-test.
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Most of the evidence available suggests an involvement of NMDA receptors in neuronal responses to prolonged, rather than acute, nociceptive inputs [17,24,35]. However, Forman [23] recently reported that MK-801, a non-competitive antagonist of the NMDA receptor, produced significant antinociception in the hot plate test. Based on these reports we assumed as an initial hypothesis that toluene was acting as an NMDA antagonist in vivo and expected to find antinociception in our experiments. Interestingly, toluene increased, not decreased, acute nociception in a concentration-dependent manner as measured by reaction time in the hotplate test. Since this finding contradicts the original hypothesis, it is reasonable to suppose that neurotransmitter systems other than the glutamatergic are implicated in the effects of toluene in acute thermonociception. Considering that the endogenous opioid system is involved in the regulation of nociception [22], we evaluated the putative role of this system in the pronociceptive effects of toluene. The interaction between NMDA receptors and opioids is well documented. Thus, some NMDA receptor antagonists are able to block or, at least diminish some opioid effects, like the development of tolerance [31,41,43] and the magnitude of the abstinence response in morphine-treated animals [38,49]. On the other hand, NMDA receptor antagonists do not counteract opiate effects, but increase them as in the case of morphine-induced analgesia [11,14,16,44]. Based on these findings, we decided to study the putative interaction between opioids and toluene in the hot plate test. According to the results presented in experiment two, morphine administration resulted in a characteristic dose-dependent increase in all the latencies to the responses evaluated seen with analgesic drugs. The combination of toluene with several doses of morphine also resulted in an antinociceptive effect, which however, did not reach the values seen in animals treated exclusively with the opioid agonist. From the shape of the dose-response curves it could be suggested that a physiological antagonism occurred between these substances. This assumption arises from the fact that the curves of morphine effects are similar in the presence and in the absence of toluene, but emerges from a different basal level [2]. To further analyze the interaction between toluene and the opioidergic system, we explored if a synergistic action occurred between naloxone and toluene. Hence, subeffective doses of this opioid antagonist were combined with low concentrations of toluene. The co-administration of these two compounds did not result in significant pronociceptive effects. From these results, it seems unlikely that the opioidergic system plays a direct role in the nociceptive effect of toluene. Although a complete pharmacological characterization of toluene’s actions on nociception is beyond the
scope of this work, it is important to mention that there is recent evidence that this abused solvent is able to affect other ligand-gated ion channels. In particular, Bale and co-workers [5] have shown preliminary data that toluene inhibits recombinant a4b2 and a3b2 receptor subtypes at micromolar concentrations. Since at least the a4b2 neuronal nAChR has been implicated in the analgesic effects of nicotine [4], one possibility is that toluene could produce its hyperalgesic effects through nicotine antagonism. Against this idea, however, is the finding that the selective nicotine antagonist, mecamylamine, is able to counteract nicotine-induced analgesia but does not produce hyperalgesia when administered alone [33]. Needless to say, specifically designed studies are needed to determine the possible participation of nicotine receptors in toluene’s pronociceptive effects. In view of the available evidence on the cellular mechanisms of action of toluene [5,8,9], the involvement of other ligand-gated ion channels in the pronociceptive effects of this solvent cannot be discarded. It is important to remember that no discrete neurotransmitter system operates in isolation, therefore, toluene’s effects might result from a complex combination of cellular effects. Further experiments are needed to investigate this point.
Acknowledgements This work was taken in part from the M.S. dissertation of Nayeli Pa´ez-Martı´nez and was supported by grant 30571M from Conacyt, Mexico. Authors wish to thank Isaı´ Me´ndez and Abraham Contreras for excellent technical assistance.
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Toluene increases acute thermonociception in mice Silvia Lorenia Cruz a,*, Nayeli Pa´ez-Martı´nez a, Francisco Pellicer b, Luis Antonio Salazar a, Carolina Lo´pez-Rubalcava a a
Departmento de Farmacobiologı´a, Cin6esta6 IPN, Apartado Postal 22026, 14000 Mexico D.F., Mexico b Di6isio´n de Neurociencias, Instituto Nacional de Psiquiatrı´a, Me´xico D.F., Mexico
Received 16 December 1999; received in revised form 02 November 2000; accepted 14 November 2000
Abstract Toluene is an abused solvent widely used in several commercial products. Recent evidence indicates that this solvent is a non-competitive inhibitor of NMDA receptors. Since NMDA receptors have been implicated in pain, this paper describes studies of the effects of increasing concentrations of inhaled toluene on nociception. Swiss Webster mice were exposed to toluene (500–8000 ppm) in static exposure chambers for 30 min. After completing the exposure period, animals were tested for nociception using the hot plate test. Toluene dose-dependently increased nociception as reflected by shorter latencies for the reflex, paw-lick and escape responses in toluene-treated mice with respect to their controls (animals exposed to air). In order to determine the possible role of opioids in this response, morphine (1 – 10 mg/kg) was injected before toluene inhalation. Toluene was not able to block morphine-induced antinocieption, however, it produced a shift of the morphine dose– response curve to lower effects, suggesting a physiological antagonism. No potentiation was seen when toluene was administered in combination with naloxone. Present results suggest that toluene increases nociception via neurotransmitter systems others than the glutamatergic. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Toluene; Nociception; Opioids; Hot-plate test; Morphine; Naloxone; Mice
1. Introduction Toluene is an industrial solvent present in many commercial products that is inhaled as a drug of abuse. Humans come into contact with it through occupational exposure or via its intentional inhalation for recreational purposes [18,27]. The mechanism of action of toluene is not completely understood, however, sound behavioral data indicate that this compound shares several effects with other central nervous system (CNS) depressants, including ethanol [21]. In agreement with these behavioral findings, recent evidence indicates that toluene, like other CNS depressants, affects ligandgated ion channel activity. Thus, toluene, at micromolar concentrations, acts as a reversible and potent non-competitive inhibitor of N-methyl-D-aspartic acid (NMDA) receptors, has no action on non-NMDA glutamate receptors [15], and inhibits nicotine receptors * Corresponding author. Tel.: +52-5-4832853; fax: +52-54832863. E-mail address: [email protected] (S.L. Cruz).
[5]. The experimental evidence on the effects of toluene on other ligand-gated channels is scarce and, therefore, less conclusive. According to Beckstead and co-workers, toluene at 0.5 mM enhances GABAA and glycine receptor activity [8]. Other groups, however, have not found changes in brain and cerebellum GABA contents in animals exposed to high concentrations of toluene [30]. As to the effects of toluene on 5HT3 receptors, preliminary data suggest that agonist-induced currents in recombinant 5HT3 receptors are enhanced in the presence of toluene [9]. The new evidence on the cellular actions of abused solvents, although limited, offers new insights for the investigation of the behavioral effects of these substances. For example, if toluene inhibits the glutamate NMDA receptor subtype, it would be interesting to examine whether its inhalation modifies pain perception. It is well established that excitatory amino acids play a significant role in nociceptive processes. Glutamate is an important neurotransmitter in primary sensory noci-
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ceptive neurons and is released in response to noxious stimuli [7,17,45]. Intrathecal injections of NMDA- and non-NMDA receptor agonists have pronociceptive effects [1,48]. On the other hand, both competitive and non-competitive NMDA receptor antagonists are effective in producing antinociception in models of persistent nociception [14,47] and, according to recent reports, also in acute models of pain [23]. In addition, a close relationship between glutamatergic and opioidergic systems exists. Opiates have been used for centuries for the treatment of pain. Opioid receptors are distributed throughout the CNS structures that are involved in transmission, modulation and sensation of pain [26,37]. The interaction between glutamate and opioids is complex. Pharmacological activation of the m receptor suppresses the magnitude of glutamate evoked postsynaptic currents through the presynaptic inhibition of the excitatory transmitter release [25]. NMDA antagonists enhance opioid antinociception [44], prevent the development of tolerance to morphine-induced analgesia and attenuate morphine withdrawal in several experimental models [10,31,32,39,43]. Thus, the relationship between NMDA and opioid receptors depends on the time of opioid exposure, previous exposure to opioid agonists, the responses under study and the relative doses of the compounds used. Based on these findings, the purpose of the present investigation was to study the acute effects of toluene on nociception in the hot plate test. The first objective was to establish a dose-response relationship for toluene effects on nociception. The second goal was to determine if an interaction between toluene and the opioidergic system existed under the circumstances of this study.
2. Material and methods
2.1. Animals Male Swiss –Webster mice (20 – 30 g) bred in our animal facilities were used in this study. Mice were housed 10 to a cage and had free access to food and water throughout the experiments. Colonies and experimental rooms were maintained under controlled light – dark cycle conditions (12:12 h, lights on at 07:00 h) with a temperature of 22 – 24°C. Testing was routinely performed between 09:00 and 15:00 h and independent groups of mice were used for each experimental condition. All procedures were performed in accordance to the regulations of the Ethics Committee of the International Association for the Study of Pain [50], and were approved by our institutional ethics committee.
2.2. Drugs Toluene 99.8% HPLC degree and naloxone hydrochloride were purchased from Sigma Chemicals Co. (St. Louis, MO). Morphine hydrochloride was obtained from Merck (Darmstadt, Germany) via the Mexican Ministry of Health. Opioids were dissolved in saline solution and injected i.p. in a volume of 0.1 ml/10 g.
2.3. Inhalation exposures Mice were exposed to air or toluene in a 29-liter static exposure chamber. In this closed system a known amount of toluene was completely volatilized to generate a concentration of 500, 1000, 2000, 4000, 6000 or 8000 ppm. The exposure chamber consisted of a chromatographic cylindrical jar covered with an acrylic lid that had injection ports and a fan that projected into the chamber above a stainless steel mesh platform [34,46]. For toluene exposure, mice were placed in the bottom of the chamber, the lid was replaced and a pre-determined amount of toluene was injected onto a filter paper located on the wire mesh. The fan was turned on within 5 s of the solvent injection to volatilize the solvent and to produce the desired concentration. Nominal vapor concentrations were confirmed with a single-wave length infrared spectrometer (Miran 1A Foxboro Analytical).
2.4. Adaptation sessions In order to diminish the influence of stress, all mice were allowed to acclimate on two consecutive days by placing them in the exposure chambers (fans functioning) for 30 min. On the third day, animals were exposed to toluene or air for 30 min and tested immediately after for nociception or motor coordination.
2.5. Hot plate test Each mouse was introduced into a glass cylinder (20 cm diameter and 25 cm height) placed at the center of a metal plate adjusted at 539 0.5°C. Within several seconds, the animal displayed three specific responses evoked by the thermal stimulus: the flexor antialgesic reflex, paw lick and escape behaviors. We considered the escape behavior as the simultaneous withdrawal of the rear limbs. The latencies to the appearance of all responses were determined and the test was concluded after 35 s to prevent tissue damage. In this model, antinociception is seen as increased latencies to the responses evaluated, while increased nociception is manifested by shorter latencies [47]. Hot plate sessions were videotaped for later measurement by an observer blind to the treatment.
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2.6. Motor coordination test The effects of toluene on motor coordination were assessed using the rota-rod test. Animals were placed on a 7 cm cylinder rotating at 11 rpm and the number of falls during a 5 min session registered. Previously, the animals were trained for three consecutive trials on the rotating cylinder. For the experimental trial, animals were exposed to either air or toluene and after removal from the exposure chamber were immediately tested again on the rota-rod. The performance of each animal after air or toluene was compared to its own performance during the previous (third) training session.
2.7. Experiment I: effects of toluene on thermonociception Independent groups of animals were exposed to air or toluene (500 –8000 ppm; n =10 each) for 30 min and tested immediately after (within 5 min) in the hot plate.
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and the other was injected with saline and exposed to air. A second experimental series was established to determine the putative interaction between toluene and the opioid competitive antagonist naloxone. As in the previous experiment, control animals were injected with saline and exposed to air. The effects of low doses of naloxone (0.1 and 0.2) alone and in combination with either toluene 500 or 1000 ppm were studied on the hot plate.
2.9. Data analysis Data were analyzed with a Kruskal –Wallis one-way Analysis of Variance (ANOVA). If significant effects were observed, a Mann –Whitney U-test was used to determine differences between groups. Comparisons of the performance on the rota-rod during the last training session and after drug treatment were done by the Wilcoxon t-test. Statistical procedures were done using the Sigma Stat program (version 2.03, Jandel Scientific).
2.8. Experiment II. interaction of toluene with opiates In order to test if the effects of toluene on nociception were due to an interaction with the endogenous opioid system, the effects of toluene exposure in morphine-treated animals were studied. To this purpose, morphine (1, 3, 10 and 17 mg/kg i.p.; n = 10 each) was injected immediately before exposing the animals either to air or to a fixed concentration of toluene (4000 ppm). Two independent groups served as controls: one was injected with saline and exposed to toluene (4000 ppm)
3. Results
3.1. Experiment I: effects of toluene on nociception A 30 min exposure to toluene induced a concentration-dependent increase in nociception evaluated in the hot plate test (Fig. 1). This pronociceptive effect was manifested as shorter latencies to the appearance of the antialgesic flexor reflex, paw lick and escape responses
Fig. 1. Effects of toluene on nociception evaluated by the latencies to the appearance of the antialgesic flexor reflex, the paw-lick and the escape behaviors in mice. Independent groups of animals (n= 10, each) were exposed for 30 min to different toluene concentrations and immediately after, tested in the hot plate. Control animals (C) were exposed to air. Each point represents the mean value 9 S.E.M. *P B 0.05; Mann–Whitney U-test.
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Fig. 2. Effects of morphine (1, 3 and 10 mg/kg, i.p) alone (empty circles) and in combination with toluene (4000 ppm, filled circles) in the hot plate test. Control groups were animals injected with saline and exposed to air (S) or to 4000 ppm toluene (T). Each point represents the mean value9S.E.M. of 10 animals. *PB 0.05; Mann–Whitney U-test.
in toluene-treated animals as compared to their controls. Although no asymptotic effects were seen at the concentration range tested, the effects of 8000 ppm toluene could be considered as maximal, since it is technically impossible to observe a flexor reflex latency shorter than 1 s. It is worth mentioning that no jumps were seen during the observation period; in our experiments, the escape response consisted of the simultaneous withdrawal of both hindlimbs. As a complement of this experiment, evaluation of the effects of toluene on motor coordination revealed no significant effects at any of the concentrations tested.
In an attempt to determine if an interaction between naloxone and toluene occurred, two experiments were done. Subeffective doses/concentrations of naloxone/ toluene were tested alone and in combination in the hot plate test. When 0.1 mg/kg naloxone and 500 ppm toluene were used (Fig. 3, upper panel), no differences were found between control, toluene-treated, naloxonetreated and combined groups. Similarly, neither 1000 ppm toluene, nor 0.2 mg/kg naloxone had significant effects on any of the responses evaluated. In this case, the combination naloxone –toluene resulted in a significant decrease only in the escape response (Fig. 3, lower panel).
3.2. Experiment II: interaction of toluene with opiates Fig. 2 shows the effects of three doses of morphine (1, 3 and 10 mg/kg) alone (empty circles) and in combination with 4000 ppm toluene (filled circles) in the hot plate test. As seen in the previous experiment, toluene, at 4000 ppm, significantly decreased the latencies to all the responses evaluated as compared with those seen in control animals injected with saline and exposed to air. As expected, morphine produced antinociceptive effects in a dose-dependent fashion observed as increased latencies to the antialgesic flexor reflex, the paw lick and escape behaviors. The combined treatment of toluene with morphine also resulted in a dose-dependent increase in all the latencies to the responses evaluated, but with values that were consistently lower than those seen in animals treated with morphine alone. A higher concentration of morphine (17 mg/kg) alone, and in combination with toluene resulted in a clear analgesia in some animals that was reflected in latencies higher than the established cutoff time (35 s) making comparisons between groups impossible to establish.
4. Discussion Toluene is used in many industrial processes [3] and is voluntarily inhaled for intoxicating purposes around the world [27]. The widespread use of this substance does not correspond to our knowledge of its mechanism of action [6]. Lately, efforts have been made to study the cellular effects of abused solvents resulting in the finding that toluene affects ligand-gated ion channels. In particular, the evidence that toluene acts as a non-competitive NMDA antagonist [15] along with the documentation that NMDA antagonists possess analgesic activity, raised the question as whether or not toluene modifies nociception. We chose the hot-plate test as an experimental tool to address this issue because it allows the study of a spinally mediated response (the antialgesic flexor reflex) and two integrated behaviors (paw lick and escape) [42]. Since it is well characterized that stress interferes with nociception tests [22], in the design of the experiments, care was
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taken to minimize the influence of animal manipulation by allowing them to adapt to the exposure chamber on two consecutive days prior to the experiment. Under these conditions, toluene induced a concentration-dependent increase in nociception in the three responses evaluated. Since, as mentioned earlier, these responses are integrated at different levels of the CNS, the pronociceptive effects of toluene appear to be a general phenomenon that involves spinal and supraspinal structures. Along with the nociception experiments, we evaluated the putative disruptive effects of toluene on motor coordination in the rota-rod test, assuming that animals who were able to walk in this test would also be able to display the integrated behaviors (paw lick and escape). No significant motor impairment was seen after any of the toluene concentrations tested. These results, however, differ from those reported by Tegeris and Balster [40] who found motor impairment in mice exposed to similar toluene concentrations. These discrepancies could probably be due to differences in sensitivity in the
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models used to evaluate motor coordination. In addition, other authors [12] have pointed out that toluene, at the concentrations used in this work, increases locomotor activity. We also observed increased activity during exposure, but it did not seem to interfere with the display of the responses evaluated in the hot plate test, since all animals were able to react to the noxious stimulus more rapidly than control mice exposed to air. The mechanisms of action of abused solvents were not studied at the molecular level until recently. A few years ago, it was generally accepted that toluene effects were due to non-specific cellular changes by virtue of its actions on membrane solubility [19,28,29]. Recent investigation has focused on more specific molecular targets finding that toluene, at relevant concentrations for behavioral effects not compromising the integrity of cellular membranes, inhibits NMDA but not AMPA and kainate receptors in vitro [15]. As previously mentioned, it has been reported that competitive and noncompetitive NMDA antagonists have antinociceptive effects in several experimental paradigms [13,20,36].
Fig. 3. Upper panel: Effects of 500 ppm toluene, 0.1 mg/kg naloxone and their combination on the antialgesic flexor reflex, the paw lick and the escape behaviors. Lower panel: Effects of 1000 ppm toluene and 0.2 mg/kg naloxone alone and in combination on the three responses evaluated in the hot plate test. Control animals (white bars) were injected with saline and exposed to air. Each bar represents the mean value 9S.E.M. of 10 mice. *P B0.05; Mann–Whitnet U-test.
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Most of the evidence available suggests an involvement of NMDA receptors in neuronal responses to prolonged, rather than acute, nociceptive inputs [17,24,35]. However, Forman [23] recently reported that MK-801, a non-competitive antagonist of the NMDA receptor, produced significant antinociception in the hot plate test. Based on these reports we assumed as an initial hypothesis that toluene was acting as an NMDA antagonist in vivo and expected to find antinociception in our experiments. Interestingly, toluene increased, not decreased, acute nociception in a concentration-dependent manner as measured by reaction time in the hotplate test. Since this finding contradicts the original hypothesis, it is reasonable to suppose that neurotransmitter systems other than the glutamatergic are implicated in the effects of toluene in acute thermonociception. Considering that the endogenous opioid system is involved in the regulation of nociception [22], we evaluated the putative role of this system in the pronociceptive effects of toluene. The interaction between NMDA receptors and opioids is well documented. Thus, some NMDA receptor antagonists are able to block or, at least diminish some opioid effects, like the development of tolerance [31,41,43] and the magnitude of the abstinence response in morphine-treated animals [38,49]. On the other hand, NMDA receptor antagonists do not counteract opiate effects, but increase them as in the case of morphine-induced analgesia [11,14,16,44]. Based on these findings, we decided to study the putative interaction between opioids and toluene in the hot plate test. According to the results presented in experiment two, morphine administration resulted in a characteristic dose-dependent increase in all the latencies to the responses evaluated seen with analgesic drugs. The combination of toluene with several doses of morphine also resulted in an antinociceptive effect, which however, did not reach the values seen in animals treated exclusively with the opioid agonist. From the shape of the dose-response curves it could be suggested that a physiological antagonism occurred between these substances. This assumption arises from the fact that the curves of morphine effects are similar in the presence and in the absence of toluene, but emerges from a different basal level [2]. To further analyze the interaction between toluene and the opioidergic system, we explored if a synergistic action occurred between naloxone and toluene. Hence, subeffective doses of this opioid antagonist were combined with low concentrations of toluene. The co-administration of these two compounds did not result in significant pronociceptive effects. From these results, it seems unlikely that the opioidergic system plays a direct role in the nociceptive effect of toluene. Although a complete pharmacological characterization of toluene’s actions on nociception is beyond the
scope of this work, it is important to mention that there is recent evidence that this abused solvent is able to affect other ligand-gated ion channels. In particular, Bale and co-workers [5] have shown preliminary data that toluene inhibits recombinant a4b2 and a3b2 receptor subtypes at micromolar concentrations. Since at least the a4b2 neuronal nAChR has been implicated in the analgesic effects of nicotine [4], one possibility is that toluene could produce its hyperalgesic effects through nicotine antagonism. Against this idea, however, is the finding that the selective nicotine antagonist, mecamylamine, is able to counteract nicotine-induced analgesia but does not produce hyperalgesia when administered alone [33]. Needless to say, specifically designed studies are needed to determine the possible participation of nicotine receptors in toluene’s pronociceptive effects. In view of the available evidence on the cellular mechanisms of action of toluene [5,8,9], the involvement of other ligand-gated ion channels in the pronociceptive effects of this solvent cannot be discarded. It is important to remember that no discrete neurotransmitter system operates in isolation, therefore, toluene’s effects might result from a complex combination of cellular effects. Further experiments are needed to investigate this point.
Acknowledgements This work was taken in part from the M.S. dissertation of Nayeli Pa´ez-Martı´nez and was supported by grant 30571M from Conacyt, Mexico. Authors wish to thank Isaı´ Me´ndez and Abraham Contreras for excellent technical assistance.
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