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Evaluation of toluene dependence and cross-sensitization to diazepam
Life Sciences 72 (2003) 3023 – 3033 www.elsevier.com/locate/lifescie
Evaluation of toluene dependence and cross-sensitization to diazepam Jenny L. Wiley *, Ambuja S. Bale, Robert L. Balster Department of Pharmacology and Toxicology, Virginia Commonwealth University, P.O. Box 980613, Richmond, VA 23298-0613, USA Received 14 October 2002; accepted 7 January 2003
Abstract Acute effects of the abused inhalant toluene resemble those of CNS depressant drugs. Since abuse of toluene involves repeated use, the purpose of the present study was to evaluate the effects of repeated or continuous exposure to toluene and to compare these effects to those of other inhalants and depressants. In experiment 1, ICR mice exposed continuously to 250 ppm toluene via inhalation for four days developed mild dependence upon termination that was characterized by an increase in severity of handling-induced convulsions. However, administration of the convulsants, N-methyl-D-aspartate (NMDA) or pentylenetetrazole (PTZ), did not differentially affect toluene- vs. air-exposed mice. In experiment 2, CFW mice (but not ICR mice) developed cross-sensitization to the initial locomotor stimulatory effects of toluene following four days of injections with 10 mg/kg/day diazepam. Previous findings have shown that 1,1,1-trichloroethane (TCE) produced robust dependence and cross-sensitization to diazepam’s locomotor effects when tested under similar conditions. The present results suggest that the dependence and cross-sensitization with diazepam produced by toluene are milder than those induced by TCE. Further, these studies add to increasing evidence that abused inhalants do not have identical pharmacological effects. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Cross-sensitization; Dependence; Diazepam; Locomotion; Toluene; Withdrawal
Introduction Toluene, a volatile hydrocarbon solvent, is contained in numerous industrial and household products, including glues, spray paints, and nail polish remover. Exposure to large concentrations of * Corresponding author. Tel.: +1-804-828-2067; fax: +1-804-828-2117. E-mail address: [email protected] (J.L. Wiley). 0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0024-3205(03)00233-9
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toluene may occur through accidental means (e.g., industrial accidents) or through intentional inhalation for the purpose of ‘‘getting high.’’ Toluene abuse occurs primarily among middle-school aged children who may move on to abuse of other drugs as they enter high school [10]. In humans, one of the primary targets of toluene is the central nervous system (CNS) where acutely it produces both excitatory and depressant behavioral effects similar to those produced by ethanol [2]. In rodent models, the acute effects of toluene also resemble those of ethanol and other CNS depressants (see Ref. [14] for a review) and include biphasic motor effects [5,6,33], anxiolysis [7,34], anticonvulsant effects [34], and ethanol-like discriminative stimulus effects [27]. Since inhalant abuse, by definition, involves chronic use, investigation of the effects of repeated administration of inhalants is important. While repeated administration of CNS depressants may induce physical dependence and produce a well-characterized withdrawal syndrome in both humans and animals [12,19,20], demonstration of physical dependence to toluene and other inhalants following repeated or chronic exposure in humans has been difficult [10]. Similarly, few animal studies have examined physical dependence to inhalants. In one such study it was found that termination of a period of four days of continuous inhalation of 1,1,1-trichloroethane (TCE) resulted in a withdrawal syndrome characterized by handlinginduced seizures [13]. The purpose of Experiment 1 was to evaluate the physical dependence liability of toluene using a similar protocol. In addition to assessing handling-induced convulsions in untreated toluene-withdrawn mice, however, we also examined the effects in these mice of pharmacological challenge with convulsants, pentylenetetrazole (PTZ) and N-methyl-D-aspartate (NMDA). These drugs have action as a g-amino butyric acid (GABA) receptor antagonist and NMDA glutamate receptor agonist, respectively. In contrast, CNS depressant effects such as those observed with acute exposure to solvents may be produced by agonism at GABA receptors or antagonism at NMDA glutamate receptors [1,32]. In particular, we hypothesized that toluene may have action at NMDA receptors based upon our previous observation that toluene partially substituted for the noncompetitive NMDA antagonist phencyclidine [9]. Since the withdrawal effects of drugs are often the opposite of their acute effects, we hypothesized that administration of these convulsants would exacerbate any withdrawal-related seizures. A second phenomenon that occurs following repeated administration of drugs of abuse, including CNS depressants, is adaptation to the initial effects of the drug. This adaptation may take the form of tolerance or sensitization. Tolerance involves decreased responsiveness to the initial effects of the drug with repeated dosing of the same drug (i.e., rightward shift of the dose-effect curve) or a different drug (cross-tolerance) whereas sensitization involves increased responsiveness to the effects of the drug with repeated dosing of the same drug (i.e., leftward shift of the dose-effect curve) or a different drug (crosssensitization). Although inhalant abusers have reported development of pronounced tolerance following chronic use [16,26], a similar degree of tolerance has not been observed in animal research. Reports of only modest or no tolerance development to the response rate decreasing effects of TCE and toluene in operant studies has been reported [22,23]. Other studies have found that sensitization develops to toluene-induced enhancement of motor activity and increases in response rates [8,18,22]. One factor that may complicate investigation of adaptation to the behavioral effects of inhalants after chronic exposure in animals is the quick clearance of inhalants from the body [3,15]. This problem is lessened with repeated injection of CNS depressant drugs that have longer half-lives. In a previous study, we demonstrated cross-sensitization to the motor-stimulating effects of TCE following repeated injection with diazepam [31]. The purpose of Experiment 2 was to determine whether repeated administration of diazepam would induce cross-tolerance/sensitization to the locomotor effects of toluene.
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Methods Subjects Male ICR (dependence and cross-sensitization studies) or CFW (cross-sensitization study) outbred strains of mice (25–32 g), purchased from Harlan (Dublin, VA), were housed in groups of six in plastic cages with wood-chip bedding. Until the time of testing, all animals were kept in a temperaturecontrolled (20–22 jC) environment with a 12-hour light-dark cycle (lights on at 7 a.m.) and received food and water ad libitum. Mice were transported to the laboratory in the same building for all testing which was carried out during the light cycle. During the dependence study, mice were housed in the exposure chamber in the laboratory with free access to food and water. Separate mice were used for testing each drug dose or inhalant concentration. The studies reported in this manuscript were carried out in accordance with guidelines published in ‘‘Guide for the care and use of laboratory animals’’ [25] and were approved by our Institutional Animal Care and Use Committee. Experiments in the ICR and CFW mice were not conducted during the same time period. Data from each of the three experiments were combined in the present manuscript based upon thematic interest rather than on systematic exploration of the effects of solvents in different strains of mice. Apparatus Vapor exposures for the dependence study were conducted in 20.8-l glass tanks fitted with Teflon lined lids. A flow regulator (R7630 series, Matheson Co., Dorsey, MD) was used to control the rate of filtered air passing through a gas dispersion tube in a 2-l flask containing toluene. Another flow regulator diluted the vapor exiting the flask with filtered air. The two flow rates were adjusted independently to maintain the 250 ppm test concentration of toluene at a flow rate of 10 l/min. Toluene concentrations in the chambers were verified through use of a single wavelength-monitoring infrared spectrometer (Miran 1A, Foxboro Analytical, North Haven, CT). Vapor exposures for the cross-sensitization studies were conducted in 29-l cylindrical jars (47 cm H 35 cm diameter; total floor space = 962 cm2) which have been described previously [22]. Briefly, vapor generation commenced when liquid toluene was injected through a port onto filter paper suspended below the sealed lid. A fan, mounted on the inside of the lid, was then turned on which volatilized and distributed the agent within the chamber. Nominal chamber concentrations did not vary by more than 10% from measured concentrations as determined by single wavelength monitoring infrared spectrometry (Miran 1A, Foxboro Analytical, North Haven, CT). Two pairs of standard photocells, mounted at right angles to each other near the bottom of the static exposure chamber, were used to measure locomotor activity. Locomotor activity was defined as the sum of the interruptions of both photocell beams (counts) during each 20-min exposure to air or toluene. A computer with Med-PC software and interfacing (Med Associates, Georgia, VT) was used to record locomotor counts. Experiment 1: Dependence Study Procedure Prior to toluene exposure, each ICR mouse was pre-tested for handling-induced seizures (as described below). Immediately following this pre-test, mice were placed in the exposure chambers in groups of
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five and exposed continuously to 250 ppm toluene or to air for four days. (250 ppm was chosen as the maximally-tolerated concentration because continuous exposure to 500 ppm toluene was found to be toxic in a pilot experiment.) Food and water were freely available in the chambers throughout exposure. Each morning during the exposure regimen mice were removed from the chamber for no more than 15 min. During this interval, they were weighed and fresh food and water were provided. Food was weighed daily in order to determine the amount of food consumption during exposure. On the morning of the fifth day, mice were removed from the chambers and tested for handling-induced convulsions. Handling-induced convulsions were scored according to the method of Evans and Balster [13] which was adapted from measures of alcohol withdrawal developed by Goldstein [17]. Mice were lifted gently by the tail, observed quickly for the appearance of tonic or clonic convulsions, given a slow 180j turn, and observed again for convulsions. Scores were given as follows: 0 = no effect in either observation 1 = tonic convulsion only when mouse is lifted and turned 2 = tonic convulsion when mouse is lifted and not turned, OR tonic-clonic convulsion when mouse is lifted and turned 3 = tonic-clonic convulsion when mouse is merely lifted 4 = tonic-clonic convulsion observable either before the mouse is lifted or after release. All mice were tested for handling-induced convulsions immediately and then at 1, 2, and 3 hours after removal. Twenty min before the third hour test mice were injected with saline, NMDA (75– 150 mg/kg), or PTZ (50 mg/kg) [n = 10 air-exposed and 10 toluene-exposed mice per dose group]. For each experiment, one individual, who was blind as to experimental treatment, scored all mice. Mice were sacrificed soon after the 3-hour observation. Experiment 2: Cross-Sensitization Study Procedure For each of the three cross-sensitization experiments described below, different groups of mice were initially exposed to a single different concentration of drug (diazepam) or solvent (toluene) on Day 1 (usually Mon) and locomotor activity was measured as number of photocell beam breaks. Each group was then injected with diazepam immediately after the locomotor session on Day 1 and every morning for the next 3 days (Tues–Thur). On the fifth day (Fri), mice were exposed to the same concentration of drug/solvent that they had received on Day 1 and locomotor activity was measured again. In the first cross-sensitization experiment, concentration-effect curve determinations with toluene were conducted in separate groups of ICR mice (n = 6 per concentration). For the initial toluene concentration-effect curve determination (Day 1), each ICR mouse was placed in the exposure chamber and exposed to air or to a single concentration of toluene. The duration of each toluene and air exposure session was 20 min, during which locomotor activity was measured. After removal from the chamber and on the next three mornings, mice were injected with 10 mg/kg diazepam. On Day 5, each mouse was re-tested with the same concentration of toluene that it had received during the initial concentrationeffect curve determination. Methods similar to those described for toluene above were used for a second and third experiments in which diazepam and toluene dose/concentration-effect curve determinations, respec-
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tively, were conducted in separate groups of CFW mice (n = 6 per dose/concentration). For the initial diazepam dose-effect curve determination (Day 1), each mouse was injected with a dose of diazepam and then placed in the exposure chamber for 20-min exposure to air. After removal from the chamber, mice in each dose group were injected with a supplemental dose of diazepam following removal from the exposure chamber such that total daily dose of diazepam equalled 10 mg/kg (e.g., vehicle-treated mice received 10 mg/kg and mice treated with 10 mg/kg diazepam received vehicle). Subsequently, they received a single 10 mg/kg dose of diazepam during the next three mornings (Days 2–4). Locomotor activity sessions were not conducted during repeated dosing with diazepam. During the morning of Day 5, a second diazepam dose-effect curve determination was conducted. Each mouse was injected with the same acute dose of diazepam that it had received during the initial dose-effect curve determination on Day 1 and was placed in the chamber for 20min exposure to air, during which locomotor activity was measured. Toluene concentration-effect curves in CFW mice were determined in an identical manner to that described for the ICR mice. Chemicals Diazepam (Schein Pharmaceuticals, Port Washington, NY) and toluene (T-324, Fisher Scientific Co., Fairlawn, NJ) were purchased commercially. The 5 mg/ml stock concentration of diazepam was diluted to desired concentrations with a vehicle of ethanol (10%), propylene glycol (40%) and sterile water (50%). NMDA (Research Biochemicals International, Natick, MA) was solubilized in distilled water and pH-adjusted with equimolar sodium hydroxide. PTZ (Sigma Chemical Co., St. Louis, MO) was dissolved in physiological saline. All diazepam, NMDA, PTZ, and vehicle injections were given i.p. in a volume of 10 ml/kg. Toluene concentrations shown in the figures and table are calculated nominal concentrations. PTZ dose was chosen based on a previous solvent dependence study with this drug. NMDA doses were chosen based upon our previous experience with this drug in drug discrimination studies and were administered in ascending order. Statistical Analysis Data for the dependence study were analyzed through the use of separate Chi-square (m2) tests at each time point or drug challenge dose. For the time course experiment, data from several groups of mice tested at 0, 1, and 2 hours after removal were combined for analysis purposes. Significant m2 tests (p < 0.05) indicated that the pattern of scores differed between toluene- and air-exposed groups. For the cross-sensitization studies, mean (F SEM) numbers of locomotor counts were calculated for the entire 20-min session. Separate split-plot ANOVAs were performed for data analysis, with time (Day 1 vs. Day 5) as the repeated factor and with toluene concentration as the between-subjects factor. When the ANOVA was significant, Tukey post hoc tests (a = 0.05) were used to compare individual means.
Results Fig. 1 shows results of the dependence study. Immediately after removal from the exposure chamber, seizure scores for toluene-exposed mice did not significantly differ from those of air-
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Fig. 1. Mice were exposed continuously for 4 days to 250 ppm toluene (right panels) or air (left panels). Top panels show the effects of toluene- or air-exposure on number of mice receiving each seizure score immediately after removal from the exposure chambers and at 1 and 2 h after removal (n = 60 at each time point for each exposure condition). Bottom panels show the effects of saline, NMDA (N; 75 – 150 mg/kg), and PTZ (P; 50 mg/kg) on number of mice receiving each score 3 h after removal from the exposure chambers (n = 10 per dose in each exposure condition). * indicates a significant difference between pattern of seizure scores for toluene-exposed mice compared to those obtained with air-exposed mice.
exposed mice (Fig. 1, top panels); however, at 1 and 2 hours following removal from the exposure chambers, seizure scores for toluene-exposed mice significantly exceeded those of air—exposed mice [m2(2) = 10.3 and 8.4 for 1 and 2 hour time points, respectively] (Fig. 1, top panels). No systematic differences in body weights or amount of food consumed between air and toluene exposed groups were noted (data not shown). At 3 hours after removal from the exposure chambers and after injection 20 min earlier with saline, NMDA, or PTZ, seizure scores of toluene- and air-exposed mice were not significantly different (Fig. 1, bottom panels). The latter results may have been due to insufficient statistical power as a result of the small number of mice that received each seizure score (i.e., low expected frequencies). We did not observe any mouse deaths following administration of either NMDA or PTZ. Fig. 2 shows locomotor counts in ICR mice across concentrations of toluene given before and after repeated administration of 10 mg/kg diazepam (Fig. 2, top panel). Toluene concentration-effect curves were similar pre- and post-diazepam treatment. A significant main effect for toluene concentration was observed [F(5,30) = 9.2, p < 0.05]; post hoc tests revealed that 8000 ppm toluene significantly
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Fig. 2. Effects of toluene on locomotor activity in ICR (top panel) and CFW (middle panel) mice before (5) and after (n) four days of repeated dosing with 10 mg/kg/day diazepam. Effects of diazepam on locomotor activity in CFW mice are also shown (bottom panel). Each point represents the mean ( F SEM) locomotor counts for 6 mice. # indicates a significant (p < 0.05) main effect for toluene concentration (i.e., the concentration significantly altered activity compared to the air condition). * indicates a significant 2-way interaction effect (i.e., post-repeated dosing mean is significantly different from the corresponding prediazepam mean). Note: a diazepam dose-effect curve for ICR mice obtained under identical conditions as described here is published in Wiley et al. [31].
decreased activity compared to air. Hence, cross-sensitization/tolerance of toluene after repeated diazepam administration did not occur in ICR mice. In contrast, significant differences were observed between toluene concentration-effect curves in CFW mice before and after diazepam treatment (significant main effects of toluene concentration and time and a significant interaction) [Fig. 2, middle panel]. Post hoc analysis of the interaction [F(5,30) = 4.3, p < 0.05] revealed that 500, 2000, and 4000 ppm toluene significantly increased locomotion following repeated diazepam administration compared to pre-diazepam activity. In CFW mice, diazepam did not produce cross-sensitization to itself (Fig. 2,
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bottom panel), although there was a trend [F(1,25) = 3.8, p = 0.06] towards a main effect for time (i.e., post-diazepam activity increased compared to pre-diazepam activity).
Discussion Four days of continuous exposure to 250 ppm toluene produced a dependence syndrome characterized by an increase in the severity of handling-induced convulsions for at least two hours following exposure termination. Although statistically significant, the magnitude of this increase was small. At three hours post-exposure, significant differences in the patterns of seizure severity between air- and toluene-exposed mice were not observed, regardless of whether or not the mice were challenged with a convulsant drug (NMDA or PTZ). These results are in contrast with those obtained following a similar exposure regimen of TCE [13]. TCE produced a pronounced withdrawal syndrome, which was worsened by administration of PTZ and attenuated by re-exposure to 2000 ppm toluene or injection with a CNS depressant (ethanol, pentobarbital, or midazolam). Several explanations of differences between the present results and previous results with TCE are possible. First, toluene and TCE do not produce identical behavioral effects in other types of procedures. For example, toluene, but not TCE, partially substituted for phencyclidine (PCP) in mice trained to discriminate PCP from saline [9]. In addition, toluene increased startle amplitudes in an acoustic startle procedure in rats whereas TCE had no effect [30]. Second, different strains of mice were used in the present study (ICR mice) and the TCE study (CFW mice). Although direct comparisons between these two strains in responsiveness to the chronic effects of inhalants have not been made, clear differences in the effects of ethanol and other CNS depressants has been demonstrated in inbred and outbred mouse strains [20,24], albeit direct comparisons between ICR and CFW mice are limited. Third, toluene may require different exposure parameters (e.g., duration, concentration) than did TCE in order to induce a more pronounced dependence. In support of this hypothesis, previous studies have shown differences in the pharmacokinetics of toluene and TCE, with toluene having a slightly slower rate of elimination [11,28]. In addition, Moser and Balster [21] reported greater sensitivity of toluene-exposed mice (vs. TCE-exposed mice) to exposure duration. Although the factors mentioned above may have affected the degree of dependence obtained with toluene in the present study, the increased severity of handling-induced convulsions within the first two hours after termination of toluene exposure is evidence that dependence did develop. In the diazepam cross-tolerance/sensitization study with ICR mice, neither sensitization nor tolerance developed to toluene’s locomotor effects following repeated dosing with diazepam. As shown previously, adaptation to diazepam’s initial suppressant effects on locomotor activity also did not develop in ICR mice tested under identical conditions [31]; hence, toluene’s effects on adaptation in this procedure did not differ from those obtained with diazepam. In contrast, four days of repeated dosing with diazepam produced sensitization to the initial locomotor stimulatory effects of TCE in ICR mice [31]. TCE and toluene were tested in the same strain as was toluene in the dependence study and under identical conditions here, suggesting that strain differences may not entirely account for behavioral differences observed between these two solvents. Although crossadaptation between toluene and CNS depressants has not been previously assessed, the present results are consistent with reports of no tolerance to the rate decreasing effects of toluene and only a nonsignificant trend toward sensitization to its rate increasing effects in mice after repeated toluene
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inhalation [22]. In rats, however, sensitization to initial increases in motor activity was observed following repeated exposure to toluene [18]. Although four to six weeks of repeated exposure to toluene was required for development of this sensitization, rats also developed sensitization to toluene’s locomotor stimulatory effects following a shorter oral dosing regimen of seven days duration [29]. In contrast to results obtained with ICR mice (and similar to those obtained with rats), CFW mice injected daily with diazepam for four days showed a trend toward sensitization to diazepam’s motor stimulatory effects as well as significant cross-sensitization to the initial motor stimulation induced by toluene. Initial concentration-effect curves for toluene in each strain were similar, although 8000 ppm toluene appeared to decrease locomotion in ICR mice to a greater extent than in CFW mice. Strain differences were most evident in comparisons of the post-diazepam concentration-effect curves. Whereas CFW mice developed sensitization to toluene’s stimulatory effects after repeated dosing with diazepam, ICR mice did not. Indeed, results here with CFW mice appear similar to those observed with TCE in ICR mice in our previous study [31]. Interestingly, previous work has demonstrated that repeated exposure to toluene also enhanced the initial locomotor activating effects of cocaine [4], although cross-sensitization between toluene and amphetamine was not observed [29]. These results suggest that adaptation to the pharmacological effects of repeated exposure to toluene and other CNS depressants may involve multiple neural mechanisms.
Conclusion In summary, continuous exposure to toluene for four days induces a brief and mild dependence syndrome characterized by an increase in severity of handling-induced convulsions. These withdrawal seizures were not differentially intensified in toluene-exposed ICR mice (vs. air-exposed mice) by administration of a convulsant (PTZ or NMDA). Development of cross-sensitization to toluene’s locomotor stimulatory effects following repeated administration of diazepam also occurred, but only in CFW (and not in ICR) mice. In contrast, TCE produced pronounced dependence and crosssensitization with diazepam [13,31]. These results suggest that there are differences in the parameters required to induce dependence and sensitization to toluene as compared to TCE. In addition, they add to increasing evidence that inhalants are not a homogeneous category of substances.
Acknowledgements This research was supported by National Institute on Drug Abuse grant DA-03112.
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Evaluation of toluene dependence and cross-sensitization to diazepam Jenny L. Wiley *, Ambuja S. Bale, Robert L. Balster Department of Pharmacology and Toxicology, Virginia Commonwealth University, P.O. Box 980613, Richmond, VA 23298-0613, USA Received 14 October 2002; accepted 7 January 2003
Abstract Acute effects of the abused inhalant toluene resemble those of CNS depressant drugs. Since abuse of toluene involves repeated use, the purpose of the present study was to evaluate the effects of repeated or continuous exposure to toluene and to compare these effects to those of other inhalants and depressants. In experiment 1, ICR mice exposed continuously to 250 ppm toluene via inhalation for four days developed mild dependence upon termination that was characterized by an increase in severity of handling-induced convulsions. However, administration of the convulsants, N-methyl-D-aspartate (NMDA) or pentylenetetrazole (PTZ), did not differentially affect toluene- vs. air-exposed mice. In experiment 2, CFW mice (but not ICR mice) developed cross-sensitization to the initial locomotor stimulatory effects of toluene following four days of injections with 10 mg/kg/day diazepam. Previous findings have shown that 1,1,1-trichloroethane (TCE) produced robust dependence and cross-sensitization to diazepam’s locomotor effects when tested under similar conditions. The present results suggest that the dependence and cross-sensitization with diazepam produced by toluene are milder than those induced by TCE. Further, these studies add to increasing evidence that abused inhalants do not have identical pharmacological effects. D 2003 Elsevier Science Inc. All rights reserved. Keywords: Cross-sensitization; Dependence; Diazepam; Locomotion; Toluene; Withdrawal
Introduction Toluene, a volatile hydrocarbon solvent, is contained in numerous industrial and household products, including glues, spray paints, and nail polish remover. Exposure to large concentrations of * Corresponding author. Tel.: +1-804-828-2067; fax: +1-804-828-2117. E-mail address: [email protected] (J.L. Wiley). 0024-3205/03/$ - see front matter D 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0024-3205(03)00233-9
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toluene may occur through accidental means (e.g., industrial accidents) or through intentional inhalation for the purpose of ‘‘getting high.’’ Toluene abuse occurs primarily among middle-school aged children who may move on to abuse of other drugs as they enter high school [10]. In humans, one of the primary targets of toluene is the central nervous system (CNS) where acutely it produces both excitatory and depressant behavioral effects similar to those produced by ethanol [2]. In rodent models, the acute effects of toluene also resemble those of ethanol and other CNS depressants (see Ref. [14] for a review) and include biphasic motor effects [5,6,33], anxiolysis [7,34], anticonvulsant effects [34], and ethanol-like discriminative stimulus effects [27]. Since inhalant abuse, by definition, involves chronic use, investigation of the effects of repeated administration of inhalants is important. While repeated administration of CNS depressants may induce physical dependence and produce a well-characterized withdrawal syndrome in both humans and animals [12,19,20], demonstration of physical dependence to toluene and other inhalants following repeated or chronic exposure in humans has been difficult [10]. Similarly, few animal studies have examined physical dependence to inhalants. In one such study it was found that termination of a period of four days of continuous inhalation of 1,1,1-trichloroethane (TCE) resulted in a withdrawal syndrome characterized by handlinginduced seizures [13]. The purpose of Experiment 1 was to evaluate the physical dependence liability of toluene using a similar protocol. In addition to assessing handling-induced convulsions in untreated toluene-withdrawn mice, however, we also examined the effects in these mice of pharmacological challenge with convulsants, pentylenetetrazole (PTZ) and N-methyl-D-aspartate (NMDA). These drugs have action as a g-amino butyric acid (GABA) receptor antagonist and NMDA glutamate receptor agonist, respectively. In contrast, CNS depressant effects such as those observed with acute exposure to solvents may be produced by agonism at GABA receptors or antagonism at NMDA glutamate receptors [1,32]. In particular, we hypothesized that toluene may have action at NMDA receptors based upon our previous observation that toluene partially substituted for the noncompetitive NMDA antagonist phencyclidine [9]. Since the withdrawal effects of drugs are often the opposite of their acute effects, we hypothesized that administration of these convulsants would exacerbate any withdrawal-related seizures. A second phenomenon that occurs following repeated administration of drugs of abuse, including CNS depressants, is adaptation to the initial effects of the drug. This adaptation may take the form of tolerance or sensitization. Tolerance involves decreased responsiveness to the initial effects of the drug with repeated dosing of the same drug (i.e., rightward shift of the dose-effect curve) or a different drug (cross-tolerance) whereas sensitization involves increased responsiveness to the effects of the drug with repeated dosing of the same drug (i.e., leftward shift of the dose-effect curve) or a different drug (crosssensitization). Although inhalant abusers have reported development of pronounced tolerance following chronic use [16,26], a similar degree of tolerance has not been observed in animal research. Reports of only modest or no tolerance development to the response rate decreasing effects of TCE and toluene in operant studies has been reported [22,23]. Other studies have found that sensitization develops to toluene-induced enhancement of motor activity and increases in response rates [8,18,22]. One factor that may complicate investigation of adaptation to the behavioral effects of inhalants after chronic exposure in animals is the quick clearance of inhalants from the body [3,15]. This problem is lessened with repeated injection of CNS depressant drugs that have longer half-lives. In a previous study, we demonstrated cross-sensitization to the motor-stimulating effects of TCE following repeated injection with diazepam [31]. The purpose of Experiment 2 was to determine whether repeated administration of diazepam would induce cross-tolerance/sensitization to the locomotor effects of toluene.
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Methods Subjects Male ICR (dependence and cross-sensitization studies) or CFW (cross-sensitization study) outbred strains of mice (25–32 g), purchased from Harlan (Dublin, VA), were housed in groups of six in plastic cages with wood-chip bedding. Until the time of testing, all animals were kept in a temperaturecontrolled (20–22 jC) environment with a 12-hour light-dark cycle (lights on at 7 a.m.) and received food and water ad libitum. Mice were transported to the laboratory in the same building for all testing which was carried out during the light cycle. During the dependence study, mice were housed in the exposure chamber in the laboratory with free access to food and water. Separate mice were used for testing each drug dose or inhalant concentration. The studies reported in this manuscript were carried out in accordance with guidelines published in ‘‘Guide for the care and use of laboratory animals’’ [25] and were approved by our Institutional Animal Care and Use Committee. Experiments in the ICR and CFW mice were not conducted during the same time period. Data from each of the three experiments were combined in the present manuscript based upon thematic interest rather than on systematic exploration of the effects of solvents in different strains of mice. Apparatus Vapor exposures for the dependence study were conducted in 20.8-l glass tanks fitted with Teflon lined lids. A flow regulator (R7630 series, Matheson Co., Dorsey, MD) was used to control the rate of filtered air passing through a gas dispersion tube in a 2-l flask containing toluene. Another flow regulator diluted the vapor exiting the flask with filtered air. The two flow rates were adjusted independently to maintain the 250 ppm test concentration of toluene at a flow rate of 10 l/min. Toluene concentrations in the chambers were verified through use of a single wavelength-monitoring infrared spectrometer (Miran 1A, Foxboro Analytical, North Haven, CT). Vapor exposures for the cross-sensitization studies were conducted in 29-l cylindrical jars (47 cm H 35 cm diameter; total floor space = 962 cm2) which have been described previously [22]. Briefly, vapor generation commenced when liquid toluene was injected through a port onto filter paper suspended below the sealed lid. A fan, mounted on the inside of the lid, was then turned on which volatilized and distributed the agent within the chamber. Nominal chamber concentrations did not vary by more than 10% from measured concentrations as determined by single wavelength monitoring infrared spectrometry (Miran 1A, Foxboro Analytical, North Haven, CT). Two pairs of standard photocells, mounted at right angles to each other near the bottom of the static exposure chamber, were used to measure locomotor activity. Locomotor activity was defined as the sum of the interruptions of both photocell beams (counts) during each 20-min exposure to air or toluene. A computer with Med-PC software and interfacing (Med Associates, Georgia, VT) was used to record locomotor counts. Experiment 1: Dependence Study Procedure Prior to toluene exposure, each ICR mouse was pre-tested for handling-induced seizures (as described below). Immediately following this pre-test, mice were placed in the exposure chambers in groups of
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five and exposed continuously to 250 ppm toluene or to air for four days. (250 ppm was chosen as the maximally-tolerated concentration because continuous exposure to 500 ppm toluene was found to be toxic in a pilot experiment.) Food and water were freely available in the chambers throughout exposure. Each morning during the exposure regimen mice were removed from the chamber for no more than 15 min. During this interval, they were weighed and fresh food and water were provided. Food was weighed daily in order to determine the amount of food consumption during exposure. On the morning of the fifth day, mice were removed from the chambers and tested for handling-induced convulsions. Handling-induced convulsions were scored according to the method of Evans and Balster [13] which was adapted from measures of alcohol withdrawal developed by Goldstein [17]. Mice were lifted gently by the tail, observed quickly for the appearance of tonic or clonic convulsions, given a slow 180j turn, and observed again for convulsions. Scores were given as follows: 0 = no effect in either observation 1 = tonic convulsion only when mouse is lifted and turned 2 = tonic convulsion when mouse is lifted and not turned, OR tonic-clonic convulsion when mouse is lifted and turned 3 = tonic-clonic convulsion when mouse is merely lifted 4 = tonic-clonic convulsion observable either before the mouse is lifted or after release. All mice were tested for handling-induced convulsions immediately and then at 1, 2, and 3 hours after removal. Twenty min before the third hour test mice were injected with saline, NMDA (75– 150 mg/kg), or PTZ (50 mg/kg) [n = 10 air-exposed and 10 toluene-exposed mice per dose group]. For each experiment, one individual, who was blind as to experimental treatment, scored all mice. Mice were sacrificed soon after the 3-hour observation. Experiment 2: Cross-Sensitization Study Procedure For each of the three cross-sensitization experiments described below, different groups of mice were initially exposed to a single different concentration of drug (diazepam) or solvent (toluene) on Day 1 (usually Mon) and locomotor activity was measured as number of photocell beam breaks. Each group was then injected with diazepam immediately after the locomotor session on Day 1 and every morning for the next 3 days (Tues–Thur). On the fifth day (Fri), mice were exposed to the same concentration of drug/solvent that they had received on Day 1 and locomotor activity was measured again. In the first cross-sensitization experiment, concentration-effect curve determinations with toluene were conducted in separate groups of ICR mice (n = 6 per concentration). For the initial toluene concentration-effect curve determination (Day 1), each ICR mouse was placed in the exposure chamber and exposed to air or to a single concentration of toluene. The duration of each toluene and air exposure session was 20 min, during which locomotor activity was measured. After removal from the chamber and on the next three mornings, mice were injected with 10 mg/kg diazepam. On Day 5, each mouse was re-tested with the same concentration of toluene that it had received during the initial concentrationeffect curve determination. Methods similar to those described for toluene above were used for a second and third experiments in which diazepam and toluene dose/concentration-effect curve determinations, respec-
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tively, were conducted in separate groups of CFW mice (n = 6 per dose/concentration). For the initial diazepam dose-effect curve determination (Day 1), each mouse was injected with a dose of diazepam and then placed in the exposure chamber for 20-min exposure to air. After removal from the chamber, mice in each dose group were injected with a supplemental dose of diazepam following removal from the exposure chamber such that total daily dose of diazepam equalled 10 mg/kg (e.g., vehicle-treated mice received 10 mg/kg and mice treated with 10 mg/kg diazepam received vehicle). Subsequently, they received a single 10 mg/kg dose of diazepam during the next three mornings (Days 2–4). Locomotor activity sessions were not conducted during repeated dosing with diazepam. During the morning of Day 5, a second diazepam dose-effect curve determination was conducted. Each mouse was injected with the same acute dose of diazepam that it had received during the initial dose-effect curve determination on Day 1 and was placed in the chamber for 20min exposure to air, during which locomotor activity was measured. Toluene concentration-effect curves in CFW mice were determined in an identical manner to that described for the ICR mice. Chemicals Diazepam (Schein Pharmaceuticals, Port Washington, NY) and toluene (T-324, Fisher Scientific Co., Fairlawn, NJ) were purchased commercially. The 5 mg/ml stock concentration of diazepam was diluted to desired concentrations with a vehicle of ethanol (10%), propylene glycol (40%) and sterile water (50%). NMDA (Research Biochemicals International, Natick, MA) was solubilized in distilled water and pH-adjusted with equimolar sodium hydroxide. PTZ (Sigma Chemical Co., St. Louis, MO) was dissolved in physiological saline. All diazepam, NMDA, PTZ, and vehicle injections were given i.p. in a volume of 10 ml/kg. Toluene concentrations shown in the figures and table are calculated nominal concentrations. PTZ dose was chosen based on a previous solvent dependence study with this drug. NMDA doses were chosen based upon our previous experience with this drug in drug discrimination studies and were administered in ascending order. Statistical Analysis Data for the dependence study were analyzed through the use of separate Chi-square (m2) tests at each time point or drug challenge dose. For the time course experiment, data from several groups of mice tested at 0, 1, and 2 hours after removal were combined for analysis purposes. Significant m2 tests (p < 0.05) indicated that the pattern of scores differed between toluene- and air-exposed groups. For the cross-sensitization studies, mean (F SEM) numbers of locomotor counts were calculated for the entire 20-min session. Separate split-plot ANOVAs were performed for data analysis, with time (Day 1 vs. Day 5) as the repeated factor and with toluene concentration as the between-subjects factor. When the ANOVA was significant, Tukey post hoc tests (a = 0.05) were used to compare individual means.
Results Fig. 1 shows results of the dependence study. Immediately after removal from the exposure chamber, seizure scores for toluene-exposed mice did not significantly differ from those of air-
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Fig. 1. Mice were exposed continuously for 4 days to 250 ppm toluene (right panels) or air (left panels). Top panels show the effects of toluene- or air-exposure on number of mice receiving each seizure score immediately after removal from the exposure chambers and at 1 and 2 h after removal (n = 60 at each time point for each exposure condition). Bottom panels show the effects of saline, NMDA (N; 75 – 150 mg/kg), and PTZ (P; 50 mg/kg) on number of mice receiving each score 3 h after removal from the exposure chambers (n = 10 per dose in each exposure condition). * indicates a significant difference between pattern of seizure scores for toluene-exposed mice compared to those obtained with air-exposed mice.
exposed mice (Fig. 1, top panels); however, at 1 and 2 hours following removal from the exposure chambers, seizure scores for toluene-exposed mice significantly exceeded those of air—exposed mice [m2(2) = 10.3 and 8.4 for 1 and 2 hour time points, respectively] (Fig. 1, top panels). No systematic differences in body weights or amount of food consumed between air and toluene exposed groups were noted (data not shown). At 3 hours after removal from the exposure chambers and after injection 20 min earlier with saline, NMDA, or PTZ, seizure scores of toluene- and air-exposed mice were not significantly different (Fig. 1, bottom panels). The latter results may have been due to insufficient statistical power as a result of the small number of mice that received each seizure score (i.e., low expected frequencies). We did not observe any mouse deaths following administration of either NMDA or PTZ. Fig. 2 shows locomotor counts in ICR mice across concentrations of toluene given before and after repeated administration of 10 mg/kg diazepam (Fig. 2, top panel). Toluene concentration-effect curves were similar pre- and post-diazepam treatment. A significant main effect for toluene concentration was observed [F(5,30) = 9.2, p < 0.05]; post hoc tests revealed that 8000 ppm toluene significantly
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Fig. 2. Effects of toluene on locomotor activity in ICR (top panel) and CFW (middle panel) mice before (5) and after (n) four days of repeated dosing with 10 mg/kg/day diazepam. Effects of diazepam on locomotor activity in CFW mice are also shown (bottom panel). Each point represents the mean ( F SEM) locomotor counts for 6 mice. # indicates a significant (p < 0.05) main effect for toluene concentration (i.e., the concentration significantly altered activity compared to the air condition). * indicates a significant 2-way interaction effect (i.e., post-repeated dosing mean is significantly different from the corresponding prediazepam mean). Note: a diazepam dose-effect curve for ICR mice obtained under identical conditions as described here is published in Wiley et al. [31].
decreased activity compared to air. Hence, cross-sensitization/tolerance of toluene after repeated diazepam administration did not occur in ICR mice. In contrast, significant differences were observed between toluene concentration-effect curves in CFW mice before and after diazepam treatment (significant main effects of toluene concentration and time and a significant interaction) [Fig. 2, middle panel]. Post hoc analysis of the interaction [F(5,30) = 4.3, p < 0.05] revealed that 500, 2000, and 4000 ppm toluene significantly increased locomotion following repeated diazepam administration compared to pre-diazepam activity. In CFW mice, diazepam did not produce cross-sensitization to itself (Fig. 2,
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bottom panel), although there was a trend [F(1,25) = 3.8, p = 0.06] towards a main effect for time (i.e., post-diazepam activity increased compared to pre-diazepam activity).
Discussion Four days of continuous exposure to 250 ppm toluene produced a dependence syndrome characterized by an increase in the severity of handling-induced convulsions for at least two hours following exposure termination. Although statistically significant, the magnitude of this increase was small. At three hours post-exposure, significant differences in the patterns of seizure severity between air- and toluene-exposed mice were not observed, regardless of whether or not the mice were challenged with a convulsant drug (NMDA or PTZ). These results are in contrast with those obtained following a similar exposure regimen of TCE [13]. TCE produced a pronounced withdrawal syndrome, which was worsened by administration of PTZ and attenuated by re-exposure to 2000 ppm toluene or injection with a CNS depressant (ethanol, pentobarbital, or midazolam). Several explanations of differences between the present results and previous results with TCE are possible. First, toluene and TCE do not produce identical behavioral effects in other types of procedures. For example, toluene, but not TCE, partially substituted for phencyclidine (PCP) in mice trained to discriminate PCP from saline [9]. In addition, toluene increased startle amplitudes in an acoustic startle procedure in rats whereas TCE had no effect [30]. Second, different strains of mice were used in the present study (ICR mice) and the TCE study (CFW mice). Although direct comparisons between these two strains in responsiveness to the chronic effects of inhalants have not been made, clear differences in the effects of ethanol and other CNS depressants has been demonstrated in inbred and outbred mouse strains [20,24], albeit direct comparisons between ICR and CFW mice are limited. Third, toluene may require different exposure parameters (e.g., duration, concentration) than did TCE in order to induce a more pronounced dependence. In support of this hypothesis, previous studies have shown differences in the pharmacokinetics of toluene and TCE, with toluene having a slightly slower rate of elimination [11,28]. In addition, Moser and Balster [21] reported greater sensitivity of toluene-exposed mice (vs. TCE-exposed mice) to exposure duration. Although the factors mentioned above may have affected the degree of dependence obtained with toluene in the present study, the increased severity of handling-induced convulsions within the first two hours after termination of toluene exposure is evidence that dependence did develop. In the diazepam cross-tolerance/sensitization study with ICR mice, neither sensitization nor tolerance developed to toluene’s locomotor effects following repeated dosing with diazepam. As shown previously, adaptation to diazepam’s initial suppressant effects on locomotor activity also did not develop in ICR mice tested under identical conditions [31]; hence, toluene’s effects on adaptation in this procedure did not differ from those obtained with diazepam. In contrast, four days of repeated dosing with diazepam produced sensitization to the initial locomotor stimulatory effects of TCE in ICR mice [31]. TCE and toluene were tested in the same strain as was toluene in the dependence study and under identical conditions here, suggesting that strain differences may not entirely account for behavioral differences observed between these two solvents. Although crossadaptation between toluene and CNS depressants has not been previously assessed, the present results are consistent with reports of no tolerance to the rate decreasing effects of toluene and only a nonsignificant trend toward sensitization to its rate increasing effects in mice after repeated toluene
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inhalation [22]. In rats, however, sensitization to initial increases in motor activity was observed following repeated exposure to toluene [18]. Although four to six weeks of repeated exposure to toluene was required for development of this sensitization, rats also developed sensitization to toluene’s locomotor stimulatory effects following a shorter oral dosing regimen of seven days duration [29]. In contrast to results obtained with ICR mice (and similar to those obtained with rats), CFW mice injected daily with diazepam for four days showed a trend toward sensitization to diazepam’s motor stimulatory effects as well as significant cross-sensitization to the initial motor stimulation induced by toluene. Initial concentration-effect curves for toluene in each strain were similar, although 8000 ppm toluene appeared to decrease locomotion in ICR mice to a greater extent than in CFW mice. Strain differences were most evident in comparisons of the post-diazepam concentration-effect curves. Whereas CFW mice developed sensitization to toluene’s stimulatory effects after repeated dosing with diazepam, ICR mice did not. Indeed, results here with CFW mice appear similar to those observed with TCE in ICR mice in our previous study [31]. Interestingly, previous work has demonstrated that repeated exposure to toluene also enhanced the initial locomotor activating effects of cocaine [4], although cross-sensitization between toluene and amphetamine was not observed [29]. These results suggest that adaptation to the pharmacological effects of repeated exposure to toluene and other CNS depressants may involve multiple neural mechanisms.
Conclusion In summary, continuous exposure to toluene for four days induces a brief and mild dependence syndrome characterized by an increase in severity of handling-induced convulsions. These withdrawal seizures were not differentially intensified in toluene-exposed ICR mice (vs. air-exposed mice) by administration of a convulsant (PTZ or NMDA). Development of cross-sensitization to toluene’s locomotor stimulatory effects following repeated administration of diazepam also occurred, but only in CFW (and not in ICR) mice. In contrast, TCE produced pronounced dependence and crosssensitization with diazepam [13,31]. These results suggest that there are differences in the parameters required to induce dependence and sensitization to toluene as compared to TCE. In addition, they add to increasing evidence that inhalants are not a homogeneous category of substances.
Acknowledgements This research was supported by National Institute on Drug Abuse grant DA-03112.
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